Anti- gprc5d antibodies, bispecific antigen binding molecules that bind gprc5d and cd3, and uses thereof

ABSTRACT

Provided herein are antibodies that specifically bind to GPRC5D. Also described are related polynucleotides capable of encoding the provided GPRC5D-specific antibodies or antigen-binding fragments, cells expressing the provided antibodies or antigen-binding fragments, as well as associated vectors and detectably labeled antibodies or antigen-binding fragments. In addition, methods of using the provided antibodies are described. For example, the provided antibodies may be used to diagnose, treat, or monitor GPRC5D-expressing cancer progression, regression, or stability; to determine whether or not a patient should be treated for cancer; or to determine whether or not a subject is afflicted with GPRC5D-expressing cancer and thus may be amenable to treatment with a GPRC5D-specific anti-cancer therapeutic, such as the multispecific antibodies against GPRC5D and CD3 described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of U.S.patent application Ser. No. 16/779,713, filed 3 Feb. 2020, which is adivisional of U.S. patent application Ser. No. 15/655,086 filed 20 Jul.2017, now U.S. Pat. No. 10,562,968 issued 18 Feb. 2020, which claims thebenefit of U.S. Provisional Application Ser. No. 62/364,811 filed 20Jul. 2016, each of which is hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jan. 20, 2023, isnamed 258199.060121_PRD3422USCNT1_SL.xml and is 94,447 bytes in size.

TECHNICAL FIELD

The disclosure provided herein relates to monoclonal antibodies thatspecifically bind G protein-coupled receptor class C group 5 member D(GPRC5D), multispecific antibodies specifically bind GPRC5D and clusterdeterminant 3 (CD3), and methods of producing and using the describedantibodies.

BACKGROUND

Multiple myeloma (MM) is the second most common hematological malignancyand constitutes 2% of all cancer deaths. MM is a heterogenous diseaseand caused by mostly by chromosome translocations inter alia t(11; 14),t(4; 14), t(8;14), del(13), del(17) (Drach et al., (1998) Blood92(3):802-809; Gertz et al., (2005) Blood 106(8):2837-2840; Facon etal., (2001) Blood 97(6): 1566-1571). MM-affected patients may experiencea variety of disease-related symptoms due to, bone marrow infiltration,bone destruction, renal failure, immunodeficiency, and the psychosocialburden of a cancer diagnosis. As of 2006, the 5-year relative survivalrate for MM was approximately 34% highlighting that MM is adifficult-to-treat disease where there are currently no curativeoptions.

G-protein coupled receptor, class C, group 5, member D (GPRC5D) is anorphan, atypical, class C GPCR first identified in 2001 (Branner-Osborneet al. Biochim Biophys Acta. 1518(3):237-248, 2001). GPRC5D and othergroup 5 GPCRs have unusually short amino-terminal domains for class Creceptors, and are therefore, predicted to be conformationally similarto class A receptors. In this regard they are unique, with sequencehomology to class C GPCRs and predicted structural topology comparableto class A receptors. Functional consequence of GPRC5D activation hasnot been described and the ligand remains unknown. The gene has threeexons and is located on chromosome 12p13.3 in humans. GPRC5D receptor ishighly conserved among various species and shares 92% identity withcynomolgus monkey GPRC5D.

GPRC5D mRNA is predominantly expressed in all malignant plasma cellsfrom MM patients (Atamaniuk J A et al. Eur J Clin Invest 42(9) 953-960;2012; Frigyesi-blood and Cohen, et al. Hematology 18(6): 348-35; 2013).GPRC5D expression is variable among the patients and correlate well withplasma cell burden and genetic aberrations such as Rb-1 deletion(Atamaniuk J A et al. Eur J Clin Invest 42(9) 953-960; 2012).

This exclusive expression of GPRC5D on the plasma-cell lineagedesignates it as an ideal target for antimyeloma antibodies.

SUMMARY

Provided herein are antibodies that specifically bind to GPRC5D andantigen-binding fragments thereof. Also described are relatedpolynucleotides capable of encoding the provided GPRC5D-specificantibodies and antigen-binding fragments, cells expressing the providedantibodies and antigen-binding fragments, as well as associated vectorsand detectably labeled antibodies and antigen-binding fragments. Inaddition, methods of using the provided antibodies and antigen-bindingfragments are described. For example, the GPRC5D-specific antibodies andantigen-binding fragments may be used to diagnose or monitorGPRC5D-expressing cancer progression, regression, or stability; todetermine whether or not a patient should be treated for cancer; or todetermine whether or not a subject is afflicted with GPRC5D-expressingcancer and thus may be amenable to treatment with a GPRC5D-specificanti-cancer therapeutic, such as the multispecific antibodies againstGPRC5D and CD3 described herein.

Further provided herein are multispecific antibodies thatimmunospecifically bind to GPRC5D and CD3 and multispecificantigen-binding fragments thereof. Also described are relatedpolynucleotides capable of encoding the providedGPRC5D×CD3-multispecific antibodies, cells expressing the providedantibodies, as well as associated vectors and detectably labeledmultispecific antibodies. In addition, methods of using the providedmultispecific antibodies are described. For example, theGPRC5D×CD3-multispecific antibodies may be used to diagnose or monitorGPRC5D-expressing cancer progression, regression, or stability; todetermine whether or not a patient should be treated for cancer; or todetermine whether or not a subject is afflicted with GPRC5D-expressingcancer and thus may be amenable to treatment with a GPRC5D-specificanti-cancer therapeutic, such as the GPRC5D×CD3-multispecific antibodiesdescribed herein.

GPRC5D-Specific Antibodies

Described herein are isolated antibodies and antigen-binding fragmentsspecific for GPRC5D. In some embodiments, the GPRC5D-specific antibodiesand antigen-binding fragments bind human GPRC5D. In some embodiments,the GPRC5D-specific antibodies and antigen-binding fragments bind humanGPRC5D and cynomolgus monkey GPRC5D. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments bind to one ormore residues of a polypeptide having the amino acid sequence of SEQ IDNO. 22. This GPRC5D-specific antibody or antigen-binding fragment mayinduce ADCC in vitro with an EC₅₀ of 28 nM or less.

Table 1 provides a summary of examples of some GPRC5D-specificantibodies described herein:

TABLE 1 CDR sequences of mAbs generated against human GPRC5D ID HC-CDR1HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 GC5B81 SYAIS GIIPIFGTANYAQKFQGESRWRGYKLD RASQSISSYLN AASSLQS QQSYSTPLT (SEQ ID NO 1) (SEQ ID NO 5)(SEQ ID NO 9) (SEQ ID NO 13) (SEQ ID NO 16) (SEQ ID NO 19) GC5B465 NYWMSGISYSGGSKYYASSVKG AAFDFGRRAVRLD RASQSISSYLN AASSLQS QQSYSTPLT GC5B597(SEQ ID NO 2) (SEQ ID NO 6) (SEQ ID NO 10) (SEQ ID NO 13) (SEQ ID NO 16)(SEQ ID NO 19) GC5B483 SYFIG IIYPGKSDTRYSPSFQG VYSFGGRHKALFDYRASQSVSSYLA DASNRAT QQRSNWPLT GC5B599 (SEQ ID NO 3) (SEQ ID NO 7)(SEQ ID NO 11) (SEQ ID NO 14) (SEQ ID NO 17) (SEQ ID NO 20) GC5B596GYTMN LINPYNSDTNYAQKLQG VALRVALDY KASQNVATHVG SASYRYS QQYNRYPYT(SEQ ID NO 4) (SEQ ID NO 8) (SEQ ID NO 12) (SEQ ID NO 15) (SEQ ID NO 18)(SEQ ID NO 21) GC5B382 DYGMH AIKYSGGSTYYADSVKG RAESGPGLDY KSSQSVLYSSNNKWASTRES QQYYSTPLT (SEQ ID NO 61) (SEQ ID NO 67) (SEQ ID NO 72)NYLA (SEQ ID  (SEQ ID NO 78) (SEQ ID NO 80) NO 98) GC5B379 NYWMSGISYSGGSKYYADSVKG AAWDFGRRAVRLDY RASQSISSYLN AASSLQS QQSYSTPLT(SEQ ID NO 2) (SEQ ID NO 28) (SEQ ID NO 30) (SEQ ID NO 13)(SEQ ID NO 16) (SEQ ID NO 19) GC5B373 SYWIG IIYPGDSDTRYSPSFQGIGFYGRSFRIFDY RASQSVSSYLA DASNRAT QQRSNWPLT (SEQ ID NO 27)(SEQ ID NO 29) (SEQ ID NO 73) (SEQ ID NO 14) (SEQ ID NO 17)(SEQ ID NO 20) GC5B376 SYWIG IIYPGDSDTRYSPSFQG VYSFGGRHKALFDYRASQSVSSYLA DASNRAT QQRSNWPLT (SEQ ID NO 27) (SEQ ID NO 29)(SEQ ID NO 11) (SEQ ID NO 14) (SEQ ID NO 17) (SEQ ID NO 20) GC5B385GYAMS AISGSGGSTYYADSVKG VDRSFGRSRYTLDY RASQSVSSYLA DASNRAT QQRSNWPLT(SEQ ID NO 62) (SEQ ID NO 68) (SEQ ID NO 74) (SEQ ID NO 14)(SEQ ID NO 17) (SEQ ID NO 20) GC5B370 SYGIS GIIPIFGNINYAQKFQGVSRRFKRFAYYFDY KSSQSVLYSSNNK WASTRES QQYYSTPLT GC5B598 (SEQ ID NO 63)(SEQ ID NO 69) (SEQ ID NO 75) NYLA (SEQ ID  (SEQ ID NO 78)(SEQ ID NO 80) NO 98) GC5B602 GYSFTGYTMN LINPYNGDTN VALRVALDYKASQNVATHVG SASYRYS QQYNRYPYT (SEQ ID NO 64) (SEQ ID NO 70)(SEQ ID NO 12) (SEQ ID NO 15) (SEQ ID NO 18) (SEQ ID NO 21) GC5B603SYAMS AISGSGGSTYYADSVKG SNFLPVVFDY RASQSVRKSLA TASNRAT QQYFRAPIT(SEQ ID NO 65) (SEQ ID NO 68) (SEQ ID NO 76) (SEQ ID NO 95)(SEQ ID NO 79) (SEQ ID NO 81) GC5B601 GFSLTSYNVH VIWAGGSTNYNSALMSDGIRLRFAY KASQNVATHVG SASYRYS QQYNRYPYT (SEQ ID NO 66) (SEQ ID NO 71)(SEQ ID NO 77) (SEQ ID NO 15) (SEQ ID NO 18) (SEQ ID NO 21)

In some embodiments are provided a GPRC5D-specific antibody, or anantigen-binding fragment thereof, comprising a heavy chain comprising aCDR1, a CDR2, and a CDR3 of any one of the antibodies described inTable 1. In some embodiments are provided a GPRC5D-specific antibody, oran antigen-binding fragment thereof, comprising a heavy chain comprisinga CDR1, a CDR2, and a CDR3 of any one of the antibodies described inTable 1 and a light chain comprising a CDR1, a CDR2, and a CDR3 of anyone of the antibodies described in Table 1. In some embodimentsdescribed herein, the GPRC5D-specific antibody or antigen-bindingfragment thereof competes for binding to GPRC5D with an antibody orantigen-binding comprising a heavy chain comprising a CDR1, a CDR2, anda CDR3 of any one of the antibodies described in Table 1 and a lightchain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodiesdescribed in Table 1.

The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 inhumans. They share more than 95% homology in the amino acid sequences ofthe Fc regions but show major differences in the amino acid compositionand structure of the hinge region. The Fc region mediates effectorfunctions, such as antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC). In ADCC, the Fc region of anantibody binds to Fc receptors (FcgRs) on the surface of immune effectorcells such as natural killers and macrophages, leading to thephagocytosis or lysis of the targeted cells. In CDC, the antibodies killthe targeted cells by triggering the complement cascade at the cellsurface. The antibodies described herein include antibodies with thedescribed features of the variable domains in combination with any ofthe IgG isotypes, including modified versions in which the Fc sequencehas been modified to effect different effector functions.

For many applications of therapeutic antibodies, Fc-mediated effectorfunctions are not part of the mechanism of action. These Fc-mediatedeffector functions can be detrimental and potentially pose a safety riskby causing off-mechanism toxicity. Modifying effector functions can beachieved by engineering the Fc regions to reduce their binding to FcgRsor the complement factors. The binding of IgG to the activating (FcgRI,FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory (FcgRIIb) FcgRs or thefirst component of complement (C1q) depends on residues located in thehinge region and the CH2 domain. Mutations have been introduced in IgG1,IgG2 and IgG4 to reduce or silence Fc functionalities. The antibodiesdescribed herein may include these modifications.

In one embodiment, the antibody comprises an Fc region with one or moreof the following properties: (a) reduced effector function when comparedto the parent Fc; (b) reduced affinity to Fcg RI, Fcg RIIa, Fcg RIIb,Fcg RIIIb and/or Fcg RIIIa, (c) reduced affinity to FcgRI (d) reducedaffinity to FcgRIIa (e) reduced affinity to FcgRIIb, (f) reducedaffinity to Fcg RIIIb or (g) reduced affinity to FcgRIIIa.

In some embodiments, the antibodies or antigen-binding fragments areIgG, or derivatives thereof, e.g., IgG1, IgG2, IgG3, and IgG4 isotypes.In some embodiments wherein the antibody has an IgG1 isotype, theantibody contains L234A, L235A, and/or K409R substitution(s) in its Fcregion. In some embodiments wherein the antibody has an IgG4 isotype,the antibody contains S228P, L234A, and L235A substitutions in its Fcregion. The antibodies described herein may include these modifications.

In addition to the described GPRC5D-specific antibodies andantigen-binding fragments, also provided are polynucleotide sequencescapable of encoding the described antibodies and antigen-bindingfragments. Vectors comprising the described polynucleotides are alsoprovided, as are cells expressing the GPRC5D-specific antibodies orantigen-binding fragments provided herein. Also described are cellscapable of expressing the disclosed vectors. These cells may bemammalian cells (such as 293F cells, CHO cells), insect cells (such asSf7 cells), yeast cells, plant cells, or bacteria cells (such as E.coli). The described antibodies may also be produced by hybridoma cells.

Methods of Using GPRC5D-Specific Antibodies

Methods of using the described GPRC5D-specific antibodies orantigen-binding fragments are also disclosed. Particular antibodies foruse in the methods discussed in this section include those with the setof CDRs described for antibodies in Table 1. For example, theseantibodies or antigen-binding fragments may be useful in treatingcancer, by interfering with GPRC5D-receptor interactions or where theantibody is conjugated to a toxin, so targeting the toxin to theGPRC5D-expressing cancer. Further, these antibodies or antigen-bindingfragments may be useful for detecting the presence of GPRC5D in abiological sample, such as blood or serum; for quantifying the amount ofGPRC5D in a biological sample, such as blood or serum; for diagnosingGPRC5D-expressing cancer; determining a method of treating a subjectafflicted with cancer; or monitoring the progression ofGPRC5D-expressing cancer in a subject. In some embodiments,GPRC5D-expressing cancer may be a lymphoma, such as multiple myeloma(MM). The described methods may be carried out before the subjectreceives treatment for GPRC5D-expressing cancer, such as treatment witha multispecific antibody against GPRC5D and CD3. Furthermore, thedescribed methods may be carried out after the subject receivestreatment for GPRC5D-expressing cancer, such as treatment with amultispecific antibody against GPRC5D and CD3 described herein.

The described methods of detecting GPRC5D in a biological sample includeexposing the biological sample to one or more of the GPRC5D-specificantibodies or antigen-binding fragments described herein.

The described methods of diagnosing GPRC5D-expressing cancer in asubject also involve exposing the biological sample to one or more ofthe GPRC5D-specific antibodies or antigen-binding fragments describedherein; however, the methods also include quantifying the amount ofGPRC5D present in the sample; comparing the amount of GPRC5D present inthe sample to a known standard or reference sample; and determiningwhether the subject's GPRC5D levels fall within the levels of GPRC5Dassociated with cancer.

Also described herein are methods of monitoring GPRC5D-expressing cancerin a subject. The described methods include exposing the biologicalsample to one or more of the GPRC5D-specific antibodies orantigen-binding fragments described herein; quantifying the amount ofGPRC5D present in the sample that is bound by the antibody, orantigen-binding fragment thereof; comparing the amount of GPRC5D presentin the sample to either a known standard or reference sample or theamount of GPRC5D in a similar sample previously obtained from thesubject; and determining whether the subject's GPRC5D levels areindicative of cancer progression, regression or stable disease based onthe difference in the amount of GPRC5D in the compared samples.

The samples obtained, or derived from, subjects are biological samplessuch as urine, blood, serum, plasma, saliva, ascites, circulating cells,circulating tumor cells, cells that are not tissue associated, tissues,surgically resected tumor tissue, biopsies, fine needle aspirationsamples, or histological preparations.

The described GPRC5D-specific antibodies or antigen-binding fragmentsmay be labeled for use with the described methods, or other methodsknown to those skilled in the art. For example, the antibodies describedherein, or antigen-binding fragments thereof, may be labeled with aradiolabel, a fluorescent label, an epitope tag, biotin, a chromophorelabel, an ECL label, an enzyme, ruthenium, ¹¹¹In-DOTA,¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase,alkaline phosphatase and beta-galactosidase, or poly-histidine orsimilar such labels known in the art.

GPRC5D-Specific Antibody Kits

Described herein are kits including the disclosed GPRC5D-specificantibodies or antigen-binding fragments thereof. The described kits maybe used to carry out the methods of using the GPRC5D-specific antibodiesor antigen-binding fragments provided herein, or other methods known tothose skilled in the art. In some embodiments the described kits mayinclude the antibodies or antigen-binding fragments described herein andreagents for use in detecting the presence of GPRC5D in a biologicalsample. Accordingly, the described kits may include one or more of theantibodies, or an antigen-binding fragment(s) thereof, described hereinand a vessel for containing the antibody or fragment when not in use,instructions for use of the antibody or fragment, the antibody orfragment affixed to a solid support, and/or detectably labeled forms ofthe antibody or fragment, as described herein.

GPRC5D×CD3-Multispecific Antibodies

The redirection of T-lymphocytes to MM cells expressing GPRC5D via theTCR/CD3 complex represents an attractive alternative approach. TheTCR/CD3 complex of T-lymphocytes consists of either a TCR alpha (α)/beta((β) or TCR gamma (γ)/delta (δ) heterodimer coexpressed at the cellsurface with the invariant subunits of CD3 labeled gamma (γ), delta (δ),epsilon (ε), zeta (ζ), and eta (η). Human CD3ε is described underUniProt P07766 (CD3E HUMAN). An anti CD3ε antibody described in thestate of the art is SP34 (Yang S J, The Journal of Immunology (1986)137; 1097-1100). SP34 reacts with both primate and human CD3. SP34 isavailable from Pharmingen. A further anti CD3 antibody described in thestate of the art is UCHT-1 (see WO2000041474). A further anti CD3antibody described in the state of the art is BC-3 (Fred HutchinsonCancer Research Institute; used in Phase I/11 trials of GvHD, Anasettiet al., Transplantation 54: 844 (1992)). SP34 differs from UCHT-1 andBC-3 in that SP-34 recognizes an epitope present on solely the c chainof CD3 (see Salmeron et al., (1991) J. Immunol. 147: 3047) whereasUCHT-1 and BC-3 recognize an epitope contributed by both the and γchains. The sequence of an antibody with the same sequence as ofantibody SP34 is mentioned in WO2008119565, WO2008119566, WO2008119567,WO2010037836, WO2010037837 and WO2010037838. A sequence which is 96%identical to VH of antibody SP34 is mentioned in U.S. Pat. No. 8,236,308(WO2007042261).

Described herein are isolated multispecific antibodies that bind GPRC5Dand CD3 (“GPRC5D×CD3 multispecific antibodies”) and multispecificantigen-binding fragments thereof. In some embodiments an isolatedantibody, or an antigen-binding fragment thereof, that bindsimmunospecifically to GPRC5D is provided.

In some embodiments, the GPRC5D-specific arm of the multispecificantibody binds human GPRC5D and cynomolgus monkey GPRC5D. In someembodiments, the GPRC5D-specific arm of the GPRC5D×CD3-multispecificantibodies or antigen-binding fragments binds the extracellular domainof human GPRC5D. In preferred embodiments, the GPRC5D×CD3 multispecificantibody or antigen-binding fragment is a bispecific antibody orantigen-binding fragment. In some embodiments, an isolated GPRC5D×CD3bispecific antibody comprising: a) a first heavy chain (HC1); b) asecond heavy chain (HC2); c) a first light chain (LC1); and d) a secondlight chain (LC2), wherein the HC1 and the LC1 pair to form a firstantigen-binding site that immunospecifically binds GPRC5D, and the HC2and the LC2 pair to form a second antigen-binding site thatimmunospecifically binds CD3, or a GPRC5D×CD3-bispecific bindingfragment thereof is provided. In another embodiment, an isolated cellexpressing the antibody or bispecific binding fragment is provided. Insome embodiments, the GPRC5D-binding arm (or “GPRC5D-specific arm”) ofthe GPRC5D×CD3 multispecific antibody is derived from a GPRC5D antibodydescribed herein (for example, from an antibody having the CDR sequenceslisted in Table 1).

In some embodiments, the GPRC5D-specific arm of theGPRC5D×CD3-multispecific antibodies or antigen-binding fragments areIgG, or derivatives thereof. In some embodiments, the CD3-binding arm(or “CD3-specific arm”) of the GPRC5D×CD3 multispecific antibody isderived from the mouse monoclonal antibody SP34, a mouse IgG3/lambdaisotype. (K. R. Abhinandan and A. C. Martin, 2008. Mol. Immunol. 45,3832-3839). In some embodiments, the CD3-binding arm of the GPRC5D×CD3multispecific antibody comprises one VH domain and one VL domainselected from Table 2.

TABLE 2Heavy chains and light chains of the CD3-specific antibody and antigen-binding fragment. VH VL CD3B219 (SEQ ID NO: 99):CD3B219 (SEQ ID NO: 100): EVQLVESGGGLVQPGGSLRLSCAASGFTFNQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYA TYAMNVWRQAPGKGLEWVARIRSKYNNYATNWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL YYAASVKGRFTISRDDSKNSLYLQMNSLKTEGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGG DTAVYYCARHGNFGNSYVSWFAYWGQGTLGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVC VTVSSASTKGPSVFPLAPCSRSTSESTAALGLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKKTVAPTECS PSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 inhumans. They share more than 95% homology in the amino acid sequences ofthe Fc regions but show major differences in the amino acid compositionand structure of the hinge region. The Fc region mediates effectorfunctions, such as antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC). In ADCC, the Fc region of anantibody binds to Fc receptors (FcgRs) on the surface of immune effectorcells such as natural killers and macrophages, leading to thephagocytosis or lysis of the targeted cells. In CDC, the antibodies killthe targeted cells by triggering the complement cascade at the cellsurface.

For many applications of therapeutic antibodies, Fc-mediated effectorfunctions are not part of the mechanism of action. These Fc-mediatedeffector functions can be detrimental and potentially pose a safety riskby causing off-mechanism toxicity. Modifying effector functions can beachieved by engineering the Fc regions to reduce their binding to FcgRsor the complement factors. The binding of IgG to the activating (FcgRI,FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory (FcgRIIb) FcgRs or thefirst component of complement (C1q) depends on residues located in thehinge region and the CH2 domain. Mutations have been introduced in IgG1,IgG2 and IgG4 to reduce or silence Fc functionalities.

In one embodiment, the antibody comprises an Fc region with one or moreof the following properties: (a) reduced effector function when comparedto the parent Fc; (b) reduced affinity to Fcg RI, Fcg RIIa, Fcg RIIb,Fcg RIIIb and/or Fcg RIIIa, (c) reduced affinity to FcgRI (d) reducedaffinity to FcgRIIa (e) reduced affinity to FcgRIIb, (f) reducedaffinity to Fcg RIIIb or (g) reduced affinity to FcgRIIIa.

In some embodiments, the CD3-specific antibody or antigen-bindingfragment from which the CD3-specific arm of the multispecific antibodyis derived is IgG, or a derivative thereof. In some embodiments, theCD3-specific antibody or antigen-binding fragment from which theCD3-specific arm of the multispecific antibody is derived is IgG1, or aderivative thereof. In some embodiments, for example, the Fc region ofthe CD3-specific IgG1 antibody from which the CD3-binding arm is derivedcomprises L234A, L235A, and F405L substitutions in its Fc region. Insome embodiments, the CD3-specific antibody or antigen-binding fragmentfrom which the CD3-specific arm of the multispecific antibody is derivedis IgG4, or a derivative thereof. In some embodiments, for example, theFc region of the CD3-specific IgG4 antibody from which the CD3-bindingarm is derived comprises S228P, L234A, L235A, F405L, and R409Ksubstitutions in its Fc region. In some embodiments, the CD3-specificantibody or antigen-binding fragment from which the CD3-specific arm ofthe multispecific antibody is derived binds CD3ε on primary human Tcells and/or primary cynomolgus T cells. In some embodiments, theCD3-specific antibody or antigen-binding fragment from which theCD3-specific arm of the multispecific antibody is derived activatesprimary human CD4+ T cells and/or primary cynomolgus CD4+ T cells.

In addition to the described GPRC5D×CD3-multispecific antibodies, alsoprovided are polynucleotide sequences capable of encoding the describedGPRC5D×CD3-multispecific antibodies. In some embodiments, an isolatedsynthetic polynucleotide encoding the HC1, the HC2, the LC1 or the LC2of the GPRC5D×CD3 bispecific antibody or bispecific binding fragment isprovided. Vectors comprising the described polynucleotides are alsoprovided, as are cells expressing the GPRC5D×CD3-multispecificantibodies provided herein. Also described are cells capable ofexpressing the disclosed vectors. These cells may be mammalian cells(such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeastcells, plant cells, or bacteria cells (such as E. coli). The describedantibodies may also be produced by hybridoma cells. In some embodiments,methods for generating the GPRC5D×CD3 bispecific antibody or bispecificbinding fragment by culturing cells is provided.

Further provided herein are pharmaceutical compositions comprising theGPRC5D×CD3 multispecific antibodies or antigen-binding fragments and apharmaceutically acceptable carrier.

Methods of Using GPRC5D×CD3-Multispecific Antibodies

Methods of using the described GPRC5D×CD3-multispecific antibodies andmultispecific antigen-binding fragments thereof are also disclosed. Forexample, the GPRC5D×CD3-multispecific antibodies and multispecificantigen-binding fragments thereof may be useful in the treatment of aGPRC5D-expressing cancer in a subject in need thereof. In someembodiments, the GPRC5D-expressing cancer is a lymphoma, such asmultiple myeloma.

The described methods of treating GPRC5D-expressing cancer in a subjectin need thereof include administering to the subject a therapeuticallyeffective amount of a described GPRC5D×CD3-multispecific antibody ormultispecific antigen-binding fragment thereof. In some embodiments, thesubject is a mammal, preferably a human. In preferred embodiments areprovided methods for treating a subject having cancer by administering atherapeutically effective amount of the GPRC5D×CD3 bispecific antibodyor bispecific antigen-binding fragment to a patient in need thereof fora time sufficient to treat the cancer.

Further provided herein are methods for inhibiting growth orproliferation of cancer cells by administering a therapeuticallyeffective amount of the GPRC5D×CD3 bispecific antibody or bispecificbinding fragment to inhibit the growth or proliferation of cancer cells.

Also provided herein are methods of redirecting a T cell to aGPRC5D-expressing cancer cell by administering a therapeuticallyeffective amount of the GPRC5D×CD3 bispecific antibody or bispecificbinding fragment to redirect a T cell to a cancer.

GPRC5D×CD3-Specific Antibody Kits

Described herein are kits including the disclosedGPRC5D×CD3-multispecific antibodies. The described kits may be used tocarry out the methods of using the GPRC5D×CD3-multispecific antibodiesprovided herein, or other methods known to those skilled in the art.

In some embodiments the described kits may include the antibodiesdescribed herein and reagents for use in treating a GPRC5D-expressingcancer. Accordingly, the described kits may include one or more of themultispecific antibodies, or a multispecific antigen-binding fragment(s)thereof, described herein and a vessel for containing the antibody orfragment when not in use, and/or instructions for use of the antibody orfragment, the antibody or fragment affixed to a solid support, and/ordetectably labeled forms of the antibody or fragment, as describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Concentration dependent binding profile of selected anti-GPRC5DmAbs against human GPRC5D and non-transfected HEK 293 cells. Three mAbs,GC5B36, GC5B168, and GC5B205 were also observed to bind to thenon-transfected (GPRC5D null) HEK293 cells.

FIG. 2 . Dose dependent binding of anti-GPCR5D×CD3 bispecific antibodiesto human GPRC5D HEK293F cells.

FIG. 3 . The binding profile of the bispecific antibodies on MM1R andH929 cells was compared with the overexpressed human GPCR5D HEK293 cellsand non-transfected HEK293 cells using FACS.

FIGS. 4A and 4B. T-cell mediated cytotoxicity and T cell activation ofanti-GPCR5D×CD3 antibodies against human GPRC5D-expressing cells.

FIGS. 5A and 5B T-cell mediated cytotoxicity and T cell activation ofanti-GPCR5D×CD3 antibodies against cyno GPRC5D-expressing cells.

FIGS. 6A and 6B. GPRC5D×CD3 Abs efficiently kill H929 cells inprophylactic NSG mice model. GCDB32 (6A) and GCDB35 (6B) resulted incomplete tumor growth inhibition at the 10 ug doses, with GCDB32 alsocapable of 100% tumor growth inhibition at the 1 ug dose.

FIGS. 7A and 7B. Comparison of potency in the absence (7A) and presence(7B) of Fc block.

FIG. 8A-8D. Binding of GCDB32 GCDB35, GCDB40 and GCDB43 to Fcγreceptors.

FIGS. 9A and 9B. FACS binding assessment of hybridoma derived mAbs.

FIGS. 10A and 10B. T-cell mediated cytotoxicity of GPRC5D×CD3 bispecificantibodies against H929 cells.

FIG. 11A-11E. GPRC5D×CD3 bispecific antibody binding to GPRC5D positive(H929, MM1R, LP1, OPM2) and negative cell lines (NALM6).

FIG. 12A-12D. GPRC5D×CD3 bispecific Abs are potent tumor inhibitors invivo. All GPRC5D×CD3 bispecific Abs (GCDB32 shown in 12A, GCDB53 shownin 12B, GCDB61 shown in 12C, and GCDB72 shown in 12D) completelyinhibited multiple myeloma cell (H929) tumor growth at the 10 ug and 1ug doses. Differentiation was observed at the 0.1 ug dose with GCDB72observed to have 80% tumor growth inhibition

FIG. 13 : GPRC5D⁺MM1.R cell lines were stained for 60 minutes withvarious concentrations of lead antibodies to measure the surface bindingprofiles (n=3). Phycoerythrin labelled human IgG4Fc was used as asecondary antibody to capture the signal (Southern Biotech, cloneHP6025). Binding is expressed as normalized geometrical meanfluorescence intensity as determined by FACS. Graphing and fitting ofdata were done in GraphPad Prism 6 using nonlinear regression withvariable slope (four parameters) and least square fit method.

FIGS. 14A and 14B. GPRC5D⁺ cell lines were stained for 60 minutes withvarious concentrations of FAB6300 and GC5M481 antibodies to measure thesurface binding profiles. Phycoerythrin labelled human IgG4Fc was usedas a secondary antibody to capture the signal (Southern Biotech, cloneHP6025). Binding is expressed as histograms (black line for the isotypeand the red line denotes for the specific GPRC5D antibody (FIG. 14A).FIG. 14B shows the binding pattern of the lead molecules on GPRC5D+multiple myeloma cell lines. Shaded, dotted line indicates isotypecontrol and the solid line indicate lead molecule binding.

FIGS. 15A and 15B. Frozen bone marrow-derived mononuclear cells from twodifferent MM patients were used to assess GPRC5D×CD3 bispecific antibodybinding, compared to IgG4 isotype control, plasma cell cytotoxicity andT-cell activation. For the cytotoxicity assay, T cells from the normalhealthy donor were exogenously added to patient BM MNC samples andincubated with four lead molecules for 48 hours. GPRC5D×CD3 bispecificantibody binds to plasma cells in a dose dependent manner to all donorsamples and the mean fluorescence intensities were recorded on theY-axis. Note the loss of live plasma cells (CD138⁺) and the concomitantupregulation of CD25 on T cells in response to GPRC5D×CD3 bispecificantibody treatment.

FIG. 16 . NSG mice were subcutaneously implanted with MM.1S humanMultiple Myeloma cells on Day 0. Human PBMC were intravenouslyinoculated on study day 7. PBS, GCDB72 (0.1 μg, 1 μg, 10 μg and 50μg/animal (equivalent to 0.005, 0.05, 0.5 and 2.5 mg/kg, respectively))and Null control antibodies were intravenously dosed on Days 15, 18, 22,24, 29, 32 and 36. Subcutaneous tumors were measured twice weekly andthe results were presented as the mean tumor volume, expressed mm³±SEMof each group. GCDB72 antibody treatment when dosed at 1 (0.05 mg/kg)significantly inhibited sc tumor growth compared to PBS (TGI=64%,p<0.0001). GCDB72 doses of 10 μg/animal (0.5 mg/kg) and 50 μg/animal(2.5 mg/kg) completely regressed tumor growth (p<0.0001). The Nullcontrol antibodies had negligible or no effect. Statistical significancewas evaluated using a 2-way ANOVA with multiple comparisons usingTukey's multiple comparisons test using Graph Pad Prism software(version 6). Differences between groups were considered significant whenthe probability value (p) was ≤0.05.

FIG. 17 . NSG were subcutaneously implanted with MM.15 human MultipleMyeloma cells on Day 0. Human PBMC were intravenously inoculated onstudy day 7. PBS, GCDB72 (0.1 μg, 1 μg, 10 μg and 50 μg/animal(equivalent to 0.005, 0.05, 0.5 and 2.5 mg/kg, respectively)) and Nullcontrol antibodies were intravenously dosed on Days 15, 18, 22, 24, 29,32 and 36. Body weight is presented as absolute body weight from startof treatment to the end of study

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of up to ±10% from the specified value, as suchvariations are appropriate to perform the disclosed methods. Unlessotherwise indicated, all numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forthused in the specification and claims are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

“Isolated” means a biological component (such as a nucleic acid, peptideor protein) has been substantially separated, produced apart from, orpurified away from other biological components of the organism in whichthe component naturally occurs, i.e., other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides andproteins that have been “isolated” thus include nucleic acids andproteins purified by standard purification methods. “Isolated” nucleicacids, peptides and proteins can be part of a composition and still beisolated if such composition is not part of the native environment ofthe nucleic acid, peptide, or protein. The term also embraces nucleicacids, peptides and proteins prepared by recombinant expression in ahost cell as well as chemically synthesized nucleic acids. An “isolated”antibody or antigen-binding fragment, as used herein, is intended torefer to an antibody or antigen-binding fragment which is substantiallyfree of other antibodies or antigen-binding fragments having differentantigenic specificities (for instance, an isolated antibody thatspecifically binds to GPRC5D is substantially free of antibodies thatspecifically bind antigens other than GPRC5D). An isolated antibody thatspecifically binds to an epitope, isoform or variant of GPRC5D may,however, have cross-reactivity to other related antigens, for instancefrom other species (such as GPRC5D species homologs).

“Polynucleotide,” synonymously referred to as “nucleic acid molecule,”“nucleotides” or “nucleic acids,” refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons.

“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications may be made to DNA andRNA; thus, “polynucleotide” embraces chemically, enzymatically ormetabolically modified forms of polynucleotides as typically found innature, as well as the chemical forms of DNA and RNA characteristic ofviruses and cells. “Polynucleotide” also embraces relatively shortnucleic acid chains, often referred to as oligonucleotides.

The meaning of “substantially the same” can differ depending on thecontext in which the term is used. Because of the natural sequencevariation likely to exist among heavy and light chains and the genesencoding them, one would expect to find some level of variation withinthe amino acid sequences or the genes encoding the antibodies orantigen-binding fragments described herein, with little or no impact ontheir unique binding properties (e.g., specificity and affinity). Suchan expectation is due in part to the degeneracy of the genetic code, aswell as to the evolutionary success of conservative amino acid sequencevariations, which do not appreciably alter the nature of the encodedprotein. Accordingly, in the context of nucleic acid sequences,“substantially the same” means at least 65% identity between two or moresequences. Preferably, the term refers to at least 70% identity betweentwo or more sequences, more preferably at least 75% identity, morepreferably at least 80% identity, more preferably at least 85% identity,more preferably at least 90% identity, more preferably at least 91%identity, more preferably at least 92% identity, more preferably atleast 93% identity, more preferably at least 94% identity, morepreferably at least 95% identity, more preferably at least 96% identity,more preferably at least 97% identity, more preferably at least 98%identity, and more preferably at least 99% or greater identity. Thepercent identity between two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The percent identity betweentwo nucleotide or amino acid sequences may e.g. be determined using thealgorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17(1988) which has been incorporated into the ALIGN program (version 2.0),using a PAM120 weight residue table, a gap length penalty of 12 and agap penalty of 4. In addition, the percent identity between two aminoacid sequences may be determined using the Needleman and Wunsch, J. Mol.Biol. 48, 444-453 (1970) algorithm.

The degree of variation that may occur within the amino acid sequence ofa protein without having a substantial effect on protein function ismuch lower than that of a nucleic acid sequence, since the samedegeneracy principles do not apply to amino acid sequences. Accordingly,in the context of an antibody or antigen-binding fragment,“substantially the same” means antibodies or antigen-binding fragmentshaving 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe antibodies or antigen-binding fragments described. Other embodimentsinclude GPRC5D specific antibodies, or antigen-binding fragments, thathave framework, scaffold, or other non-binding regions that do not sharesignificant identity with the antibodies and antigen-binding fragmentsdescribed herein, but do incorporate one or more CDRs or other sequencesneeded to confer binding that are 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to such sequences described herein. A“vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations. In some examplesprovided herein, cells are transformed by transfecting the cells withDNA.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA.

These terms also encompass translation of RNA into one or morepolypeptides, and further encompass all naturally occurringpost-transcriptional and post-translational modifications. Theexpression or production of an antibody or antigen-binding fragmentthereof may be within the cytoplasm of the cell, or into theextracellular milieu such as the growth medium of a cell culture.

The terms “treating” or “treatment” refer to any success or indicia ofsuccess in the attenuation or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement, remission, diminishing of symptoms or making the conditionmore tolerable to the patient, slowing in the rate of degeneration ordecline, making the final point of degeneration less debilitating,improving a subject's physical or mental well-being, or prolonging thelength of survival. The treatment may be assessed by objective orsubjective parameters; including the results of a physical examination,neurological examination, or psychiatric evaluations.

An “effective amount” or “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve a desired therapeutic result. A therapeutically effective amountof a GPRC5D×CD3 antibody may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the antibody to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects.

“Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE,IgM, IgD, and IgY) including various monomeric, polymeric and chimericforms, unless otherwise specified. Specifically encompassed by the term“antibody” are polyclonal antibodies, monoclonal antibodies (mAbs), andantibody-like polypeptides, such as chimeric antibodies and humanizedantibodies.

“Antigen-binding fragments” are any proteinaceous structure that mayexhibit binding affinity for a particular antigen. Antigen-bindingfragments include those provided by any known technique, such asenzymatic cleavage, peptide synthesis, and recombinant techniques. Someantigen-binding fragments are composed of portions of intact antibodiesthat retain antigen-binding specificity of the parent antibody molecule.For example, antigen-binding fragments may comprise at least onevariable region (either a heavy chain or light chain variable region) orone or more CDRs of an antibody known to bind a particular antigen.Examples of suitable antigen-binding fragments include, withoutlimitation diabodies and single-chain molecules as well as Fab, F(ab′)2,Fc, Fabc, and Fv molecules, single chain (Sc) antibodies, individualantibody light chains, individual antibody heavy chains, chimericfusions between antibody chains or CDRs and other proteins, proteinscaffolds, heavy chain monomers or dimers, light chain monomers ordimers, dimers consisting of one heavy and one light chain, a monovalentfragment consisting of the VL, VH, CL and CH1 domains, or a monovalentantibody as described in WO2007059782, bivalent fragments comprising twoFab fragments linked by a disulfide bridge at the hinge region, a Fdfragment consisting essentially of the V.sub.H and C.sub.H1 domains; aFv fragment consisting essentially of the VL and VH domains of a singlearm of an antibody, a dAb fragment (Ward et al., Nature 341, 544-546(1989)), which consists essentially of a VH domain and also calleddomain antibodies (Holt et al; Trends Biotechnol. 2003 November;21(11):484-90); camelid or nanobodies (Revets et al; Expert Opin BiolTher. 2005 January; 5(1):111-24); an isolated complementaritydetermining region (CDR), and the like. All antibody isotypes may beused to produce antigen-binding fragments. Additionally, antigen-bindingfragments may include non-antibody proteinaceous frameworks that maysuccessfully incorporate polypeptide segments in an orientation thatconfers affinity for a given antigen of interest, such as proteinscaffolds. Antigen-binding fragments may be recombinantly produced orproduced by enzymatic or chemical cleavage of intact antibodies. Thephrase “an antibody or antigen-binding fragment thereof” may be used todenote that a given antigen-binding fragment incorporates one or moreamino acid segments of the antibody referred to in the phrase.

The terms “CDR”, and its plural “CDRs”, refer to a complementaritydetermining region (CDR) of which three make up the binding character ofa light chain variable region (CDRL1, CDRL2 and CDRL3) and three make upthe binding character of a heavy chain variable region (CDRH1, CDRH2 andCDRH3). CDRs contribute to the functional activity of an antibodymolecule and are separated by amino acid sequences that comprisescaffolding or framework regions. The exact definitional CDR boundariesand lengths are subject to different classification and numberingsystems. CDRs may therefore be referred to by Kabat, Chothia, contact orany other boundary definitions. Despite differing boundaries, each ofthese systems has some degree of overlap in what constitutes the socalled “hypervariable regions” within the variable sequences. CDRdefinitions according to these systems may therefore differ in lengthand boundary areas with respect to the adjacent framework region. Seefor example Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al.,“Canonical Structures For the Hypervariable Regions of Immunoglobulins,”J. Mol. Biol. 196:901 (1987); and MacCallum et al., “Antibody-AntigenInteractions: Contact Analysis and Binding Site Topography,” J. Mol.Biol. 262:732 (1996)), each of which is hereby incorporated by referencein its entirety.

Typically, CDRs form a loop structure that can be classified as acanonical structure. The term “canonical structure” refers to the mainchain conformation that is adopted by the antigen binding (CDR) loops.From comparative structural studies, it has been found that five of thesix antigen binding loops have only a limited repertoire of availableconformations. Each canonical structure can be characterized by thetorsion angles of the polypeptide backbone. Correspondent loops betweenantibodies may, therefore, have very similar three dimensionalstructures, despite high amino acid sequence variability in most partsof the loops (Chothia et al., “Canonical Structures For theHypervariable Regions of Immunoglobulins,” J. Mol. Biol. 196:901 (1987);Chothia et al., “Conformations of Immunoglobulin Hypervariable Regions,”I 342:877 (1989); Martin and Thornton, “Structural Families in Loops ofHomologous Proteins: Automatic Classification, Modelling and Applicationto Antibodies,” J. Mol. Biol. 263:800 (1996), each of which isincorporated by reference in its entirety). Furthermore, there is arelationship between the adopted loop structure and the amino acidsequences surrounding it. The conformation of a particular canonicalclass is determined by the length of the loop and the amino acidresidues residing at key positions within the loop, as well as withinthe conserved framework (i.e., outside of the loop). Assignment to aparticular canonical class can therefore be made based on the presenceof these key amino acid residues.

The term “polypeptide” is used interchangeably with the term “protein”and in its broadest sense refers to a compound of two or more subunitamino acids, amino acid analogs or peptidomimetics. The subunits may belinked by peptide bonds. In another embodiment, the subunit may belinked by other bonds, e.g., ester, ether, etc. As used herein the term“amino acid” refers to either natural and/or unnatural or syntheticamino acids, including glycine and both the D and L optical isomers,amino acid analogs and peptidomimetics. A peptide of three or more aminoacids is commonly called an oligopeptide if the peptide chain is short.If the peptide chain is long, the peptide is commonly called apolypeptide or a protein.

“Specifically binds” or “binds specifically” or derivatives thereof whenused in the context of antibodies, or antibody fragments, representsbinding via domains encoded by immunoglobulin genes or fragments ofimmunoglobulin genes to one or more epitopes of a protein of interest,without preferentially binding other molecules in a sample containing amixed population of molecules. Typically, an antibody binds to a cognateantigen with a K_(d) of less than about 1×10⁻⁸ M, as measured by asurface plasmon resonance assay or a cell-binding assay. Phrases such as“[antigen]-specific” antibody (e.g., GPRC5D-specific antibody) are meantto convey that the recited antibody specifically binds the recitedantigen.

Polynucleotide,” synonymously referred to as “nucleic acid molecule,”“nucleotides” or “nucleic acids,” refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short nucleic acidchains, often referred to as oligonucleotides.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

As used herein, the term “host cell” can be any type of cell, e.g., aprimary cell, a cell in culture, or a cell from a cell line. In specificembodiments, the term “host cell” refers to a cell transfected with anucleic acid molecule and the progeny or potential progeny of such acell. Progeny of such a cell may not be identical to the parent celltransfected with the nucleic acid molecule, e.g., due to mutations orenvironmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome. Theterms “expression” and “production” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression or production of an antibody orantigen-binding fragment thereof may be within the cytoplasm of thecell, or into the extracellular milieu such as the growth medium of acell culture. The meaning of “substantially the same” can differdepending on the context in which the term is used. Because of thenatural sequence variation likely to exist among heavy and light chainsand the genes encoding them, one would expect to find some level ofvariation within the amino acid sequences or the genes encoding theantibodies or antigen-binding fragments described herein, with little orno impact on their unique binding properties (e.g., specificity andaffinity). Such an expectation is due in part to the degeneracy of thegenetic code, as well as to the evolutionary success of conservativeamino acid sequence variations, which do not appreciably alter thenature of the encoded protein. Accordingly, in the context of nucleicacid sequences, “substantially the same” means at least 65% identitybetween two or more sequences. Preferably, the term refers to at least70% identity between two or more sequences, more preferably at least 75%identity, more preferably at least 80% identity, more preferably atleast 85% identity, more preferably at least 90% identity, morepreferably at least 91% identity, more preferably at least 92% identity,more preferably at least 93% identity, more preferably at least 94%identity, more preferably at least 95% identity, more preferably atleast 96% identity, more preferably at least 97% identity, morepreferably at least 98% identity, and more preferably at least 99% orgreater identity. The percent identity between two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The percent identity between two nucleotide or amino acid sequences maye.g. be determined using the algorithm of E. Meyers and W. Miller,Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated intothe ALIGN program (version 2.0), using a PAM120 weight residue table, agap length penalty of 12 and a gap penalty of 4. In addition, thepercent identity between two amino acid sequences may be determinedusing the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970)algorithm.

The degree of variation that may occur within the amino acid sequence ofa protein without having a substantial effect on protein function ismuch lower than that of a nucleic acid sequence, since the samedegeneracy principles do not apply to amino acid sequences.

Accordingly, in the context of an antibody or antigen-binding fragment,“substantially the same” means antibodies or antigen-binding fragmentshaving 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe antibodies or antigen-binding fragments described. Other embodimentsinclude GPRC5D-specific antibodies, or antigen-binding fragments, thathave framework, scaffold, or other non-binding regions that do not sharesignificant identity with the antibodies and antigen-binding fragmentsdescribed herein, but do incorporate one or more CDRs or other sequencesneeded to confer binding that are 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to such sequences described herein.

The term “subject” refers to human and non-human animals, including allvertebrates, e.g., mammals and non-mammals, such as non-human primates,mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians,and reptiles. In many embodiments of the described methods, the subjectis a human.

The term “redirect” or “redirecting” as used herein refers to theability of the GPRC5D×CD3 antibody to traffic the activity of T cellseffectively, from its inherent cognate specificity toward reactivityagainst GPRC5D-expressing cells.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues (e.g., surgically resected tumor tissue,biopsies, including fine needle aspiration), isolated from a subject, aswell as fluids, cells, or tissues present within a subject. In someembodiments the sample is a biological fluid. Biological fluids aretypically liquids at physiological temperatures and may includenaturally occurring fluids present in, withdrawn from, expressed orotherwise extracted from a subject or biological source. Certainbiological fluids derive from particular tissues, organs or localizedregions and certain other biological fluids may be more globally orsystemically situated in a subject or biological source. Examples ofbiological fluids include blood, serum and serosal fluids, plasma,lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosalsecretions of the secretory tissues and organs, vaginal secretions,ascites fluids such as those associated with non-solid tumors, fluids ofthe pleural, pericardial, peritoneal, abdominal and other body cavities,fluids collected by bronchial lavage and the like. Biological fluids mayalso include liquid solutions contacted with a subject or biologicalsource, for example, cell and organ culture medium including cell ororgan conditioned medium, lavage fluids and the like. The term “sample,”as used herein, encompasses materials removed from a subject ormaterials present in a subject.

A “known standard” may be a solution having a known amount orconcentration of GPRC5D, where the solution may be a naturally occurringsolution, such as a sample from a patient known to have early, moderate,late, progressive, or static cancer, or the solution may be a syntheticsolution such as buffered water having a known amount of GPRC5D dilutedtherein. The known standards, described herein may include GPRC5Disolated from a subject, recombinant or purified GPRC5D protein, or avalue of GPRC5D concentration associated with a disease condition.

As used herein, the terms “G-protein coupled receptor family C group 5member D” and “GPRC5D” specifically include the human GPRC5D protein,for example as described in GenBank Accession No. BC069341, NCBIReference Sequence: NP_061124.1 and UniProtKB/Swiss-Prot Accession No.Q9NZD1 (see also Brauner-Osborne, H. et al. 2001, Biochim. Biophys. Acta1518, 237-248).

The term “CD3” refers to the human CD3 protein multi-subunit complex.The CD3 protein multi-subunit complex is composed to 6 distinctivepolypeptide chains. These include a CD3γ chain (SwissProt P09693), aCD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), andone CD3 ζ chain homodimer (SwissProt 20963), and which is associatedwith the T cell receptor α and β chain. The term “CD3” includes any CD3variant, isoform and species homolog which is naturally expressed bycells (including T cells) or can be expressed on cells transfected withgenes or cDNA encoding those polypeptides, unless noted.

A “GPRC5D×CD3 antibody” is a multispecific antibody, optionally abispecific antibody, which comprises two different antigen-bindingregions, one of which binds specifically to the antigen GPRC5D and oneof which binds specifically to CD3. A multispecific antibody can be abispecific antibody, diabody, or similar molecule (see for instance PNASUSA 90(14), 6444-8 (1993) for a description of diabodies). Thebispecific antibodies, diabodies, and the like, provided herein may bindany suitable target in addition to a portion of GPRC5D. The term“bispecific antibody” is to be understood as an antibody having twodifferent antigen-binding regions defined by different antibodysequences. This can be understood as different target binding butincludes as well binding to different epitopes in one target.

A “reference sample” is a sample that may be compared against anothersample, such as a test sample, to allow for characterization of thecompared sample. The reference sample will have some characterizedproperty that serves as the basis for comparison with the test sample.For instance, a reference sample may be used as a benchmark for GPRC5Dlevels that are indicative of a subject having cancer. The referencesample does not necessarily have to be analyzed in parallel with thetest sample, thus in some instances the reference sample may be anumerical value or range previously determined to characterize a givencondition, such as GPRC5D levels that are indicative of cancer in asubject. The term also includes samples used for comparative purposesthat are known to be associated with a physiologic state or diseasecondition, such as GPRC5D-expressing cancer, but that have an unknownamount of GPRC5D.

The term “progression,” as used in the context of progression ofGPRC5D-expressing cancer, includes the change of a cancer from a lesssevere to a more severe state. This may include an increase in thenumber or severity of tumors, the degree of metastasis, the speed withwhich the cancer is growing or spreading, and the like. For example,“the progression of colon cancer” includes the progression of such acancer from a less severe to a more severe state, such as theprogression from stage I to stage II, from stage II to stage III, etc.

The term “regression,” as used in the context of regression ofGPRC5D-expressing cancer, includes the change of a cancer from a moresevere to a less severe state. This could include a decrease in thenumber or severity of tumors, the degree of metastasis, the speed withwhich the cancer is growing or spreading, and the like. For example,“the regression of colon cancer” includes the regression of such acancer from a more severe to a less severe state, such as theprogression from stage III to stage II, from stage II to stage I, etc.

The term “stable” as used in the context of stable GPRC5D-expressingcancer, is intended to describe a disease condition that is not, or hasnot, changed significantly enough over a clinically relevant period oftime to be considered a progressing cancer or a regressing cancer.

The embodiments described herein are not limited to particular methods,reagents, compounds, compositions or biological systems, which can, ofcourse, vary.

GPRC5D-Specific Antibodies and Antigen-Binding Fragments

Described herein are isolated monoclonal antibodies or antigen-bindingfragments that specifically bind GPRC5D. The general structure of anantibody molecule comprises an antigen binding domain, which includesheavy and light chains, and the Fc domain, which serves a variety offunctions, including complement fixation and binding antibody receptors.

The described GPRC5D-specific antibodies or antigen-binding fragmentsinclude all isotypes, IgA, IgD, IgE, IgG and IgM, and syntheticmultimers of the four-chain immunoglobulin structure. The describedantibodies or antigen-binding fragments also include the IgY isotypegenerally found in hen or turkey serum and hen or turkey egg yolk.

The GPRC5D-specific antibodies and antigen-binding fragments may bederived from any species by recombinant means. For example, theantibodies or antigen-binding fragments may be mouse, rat, goat, horse,swine, bovine, chicken, rabbit, camelid, donkey, human, or chimericversions thereof. For use in administration to humans, non-human derivedantibodies or antigen-binding fragments may be genetically orstructurally altered to be less antigenic upon administration to a humanpatient.

In some embodiments, the antibodies or antigen-binding fragments arechimeric. As used herein, the term “chimeric” refers to an antibody, orantigen-binding fragment thereof, having at least some portion of atleast one variable domain derived from the antibody amino acid sequenceof a non-human mammal, a rodent, or a reptile, while the remainingportions of the antibody, or antigen-binding fragment thereof, arederived from a human.

In some embodiments, the antibodies are humanized antibodies. Humanizedantibodies may be chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions are those of a humanimmunoglobulin sequence. The humanized antibody may include at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin.

The antibodies or antigen-binding fragments described herein can occurin a variety of forms, but will include one or more of the antibody CDRsshown in Table 1.

Described herein are isolated antibodies and antigen-binding fragmentsthat immunospecifically bind to GPRC5D. In some embodiments, theGPRC5D-specific antibodies or antigen-binding fragments are human IgG,or derivatives thereof. While the GPRC5D-specific antibodies orantigen-binding fragments exemplified herein are human, the antibodiesor antigen-binding fragments exemplified may be chimerized.

In some embodiments are provided a GPRC5D-specific antibody, or anantigen-binding fragment thereof, comprising a heavy chain comprising aCDR1, a CDR2, and a CDR3 of any one of the antibodies described inTable 1. In some embodiments are provided a GPRC5D-specific antibody, oran antigen-binding fragment thereof, comprising a heavy chain comprisinga CDR1, a CDR2, and a CDR3 of any one of the antibodies described inTable 1 and a light chain comprising a CDR1, a CDR2, and a CDR3 of anyone of the antibodies described in Table 1.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 1, a heavychain CDR2 comprising SEQ ID NO: 5, and a heavy chain CDR3 comprisingSEQ ID NO: 9. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 1, a heavy chain CDR2 comprising SEQ ID NO: 5, a heavy chain CDR3comprising SEQ ID NO: 9, a light chain CDR1 comprising SEQ ID NO: 13, alight chain CDR2 comprising SEQ ID NO: 16, and a light chain CDR3comprising SEQ ID NO: 19. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 52. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 52 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 56. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 2, a heavychain CDR2 comprising SEQ ID NO: 6, and a heavy chain CDR3 comprisingSEQ ID NO: 10. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 2, a heavy chain CDR2 comprising SEQ ID NO: 6, a heavy chain CDR3comprising SEQ ID NO: 10, a light chain CDR1 comprising SEQ ID NO: 13, alight chain CDR2 comprising SEQ ID NO: 16, and a light chain CDR3comprising SEQ ID NO: 19.

This GPRC5D-specific antibody or antigen-binding fragment may comprisehuman framework sequences. In some embodiments, the GPRC5D-specificantibodies and antigen-binding fragments comprise a heavy chain variabledomain substantially the same as, or identical to, SEQ ID NO: 53. Insome embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 53 and a light chain variable domainsubstantially the same as, or identical to, SEQ ID NO: 56. The heavychain variable domain and light chain variable domain of antibodiesdiscussed in this paragraph are suitable for inclusion in bispecificconstructs in which one arm is an anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavychain CDR2 comprising SEQ ID NO: 7, and a heavy chain CDR3 comprisingSEQ ID NO: 11. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, a heavy chain CDR3comprising SEQ ID NO: 11, a light chain CDR1 comprising SEQ ID NO: 14, alight chain CDR2 comprising SEQ ID NO: 17, and a light chain CDR3comprising SEQ ID NO: 20. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 54. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 54 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 57. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 4, a heavychain CDR2 comprising SEQ ID NO: 8, and a heavy chain CDR3 comprisingSEQ ID NO: 12. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 4, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3comprising SEQ ID NO: 12, a light chain CDR1 comprising SEQ ID NO: 15, alight chain CDR2 comprising SEQ ID NO: 18, and a light chain CDR3comprising SEQ ID NO: 21. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 55. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 55 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 58. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 61, a heavychain CDR2 comprising SEQ ID NO: 67, and a heavy chain CDR3 comprisingSEQ ID NO: 72. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 61, a heavy chain CDR2 comprising SEQ ID NO: 67, a heavy chain CDR3comprising SEQ ID NO: 72, a light chain CDR1 comprising SEQ ID NO: 13, alight chain CDR2 comprising SEQ ID NO: 78, and a light chain CDR3comprising SEQ ID NO: 80. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 82. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 82 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 92. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 2, a heavychain CDR2 comprising SEQ ID NO: 28, and a heavy chain CDR3 comprisingSEQ ID NO: 30. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 2, a heavy chain CDR2 comprising SEQ ID NO: 28, a heavy chain CDR3comprising SEQ ID NO: 30, a light chain CDR1 comprising SEQ ID NO: 13, alight chain CDR2 comprising SEQ ID NO: 16, and a light chain CDR3comprising SEQ ID NO: 19. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 83. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 83 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 56. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavychain CDR2 comprising SEQ ID NO: 29, and a heavy chain CDR3 comprisingSEQ ID NO: 73. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 27, a heavy chain CDR2 comprising SEQ ID NO: 29, a heavy chain CDR3comprising SEQ ID NO: 73, a light chain CDR1 comprising SEQ ID NO: 14, alight chain CDR2 comprising SEQ ID NO: 17, and a light chain CDR3comprising SEQ ID NO: 20.

This GPRC5D-specific antibody or antigen-binding fragment may comprisehuman framework sequences. In some embodiments, the GPRC5D-specificantibodies and antigen-binding fragments comprise a heavy chain variabledomain substantially the same as, or identical to, SEQ ID NO: 84. Insome embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 84 and a light chain variable domainsubstantially the same as, or identical to, SEQ ID NO: 57. The heavychain variable domain and light chain variable domain of antibodiesdiscussed in this paragraph are suitable for inclusion in bispecificconstructs in which one arm is an anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavychain CDR2 comprising SEQ ID NO: 29, and a heavy chain CDR3 comprisingSEQ ID NO: 11. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 27, a heavy chain CDR2 comprising SEQ ID NO: 29 a heavy chain CDR3comprising SEQ ID NO: 11, a light chain CDR1 comprising SEQ ID NO: 14, alight chain CDR2 comprising SEQ ID NO: 17, and a light chain CDR3comprising SEQ ID NO: 20. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 85. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 85 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 57. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 62, a heavychain CDR2 comprising SEQ ID NO: 68, and a heavy chain CDR3 comprisingSEQ ID NO: 74. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 62, a heavy chain CDR2 comprising SEQ ID NO: 68, a heavy chain CDR3comprising SEQ ID NO: 74, a light chain CDR1 comprising SEQ ID NO: 14, alight chain CDR2 comprising SEQ ID NO: 17, and a light chain CDR3comprising SEQ ID NO: 20. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 86. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 86 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 57. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 63, a heavychain CDR2 comprising SEQ ID NO: 69, and a heavy chain CDR3 comprisingSEQ ID NO: 75. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 63, a heavy chain CDR2 comprising SEQ ID NO: 69, a heavy chain CDR3comprising SEQ ID NO: 75, a light chain CDR1 comprising SEQ ID NO: 13, alight chain CDR2 comprising SEQ ID NO: 78, and a light chain CDR3comprising SEQ ID NO: 80. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 87. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 87 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 92. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 64, a heavychain CDR2 comprising SEQ ID NO: 70, and a heavy chain CDR3 comprisingSEQ ID NO: 12. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 64, a heavy chain CDR2 comprising SEQ ID NO: 70, a heavy chain CDR3comprising SEQ ID NO: 12, a light chain CDR1 comprising SEQ ID NO: 15, alight chain CDR2 comprising SEQ ID NO: 18, and a light chain CDR3comprising SEQ ID NO: 21.

This GPRC5D-specific antibody or antigen-binding fragment may comprisehuman framework sequences. In some embodiments, the GPRC5D-specificantibodies and antigen-binding fragments comprise a heavy chain variabledomain substantially the same as, or identical to, SEQ ID NO: 88. Insome embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 88 and a light chain variable domainsubstantially the same as, or identical to, SEQ ID NO: 58. The heavychain variable domain and light chain variable domain of antibodiesdiscussed in this paragraph are suitable for inclusion in bispecificconstructs in which one arm is an anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 65, a heavychain CDR2 comprising SEQ ID NO: 68, and a heavy chain CDR3 comprisingSEQ ID NO: 76. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 65, a heavy chain CDR2 comprising SEQ ID NO: 68, a heavy chain CDR3comprising SEQ ID NO: 76, a light chain CDR1 comprising SEQ ID NO: 95, alight chain CDR2 comprising SEQ ID NO: 79, and a light chain CDR3comprising SEQ ID NO: 81. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 89. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 89 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 93. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain CDR1 comprising SEQ ID NO: 66, a heavychain CDR2 comprising SEQ ID NO: 71, and a heavy chain CDR3 comprisingSEQ ID NO: 77. In some embodiments, the GPRC5D-specific antibodies andantigen-binding fragments comprise a heavy chain CDR1 comprising SEQ IDNO: 66, a heavy chain CDR2 comprising SEQ ID NO: 71, a heavy chain CDR3comprising SEQ ID NO: 77, a light chain CDR1 comprising SEQ ID NO: 15, alight chain CDR2 comprising SEQ ID NO: 18, and a light chain CDR3comprising SEQ ID NO: 21. This GPRC5D-specific antibody orantigen-binding fragment may comprise human framework sequences. In someembodiments, the GPRC5D-specific antibodies and antigen-bindingfragments comprise a heavy chain variable domain substantially the sameas, or identical to, SEQ ID NO: 91. In some embodiments, theGPRC5D-specific antibodies and antigen-binding fragments comprise aheavy chain variable domain substantially the same as, or identical to,SEQ ID NO: 91 and a light chain variable domain substantially the sameas, or identical to, SEQ ID NO: 94. The heavy chain variable domain andlight chain variable domain of antibodies discussed in this paragraphare suitable for inclusion in bispecific constructs in which one arm isan anti-GPRC5D arm.

In some embodiments, the antibodies or antigen-binding fragments areIgG, or derivatives thereof, e.g., IgG1, IgG2, IgG3, and IgG4 isotypes.In some embodiments wherein the antibody is of IgG1 isotype, theantibody comprises an IgG1 Fc region (SEQ ID NO. 60).

SEQ ID NO. 60 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIn some embodiments wherein the antibody is of IgG4 isotype, theantibody contains S228P, L234A, and L235A substitutions in its Fc region(SEQ ID NO. 59).

SEQ ID NO. 59 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

The specific antibodies defined by CDR and/or variable domain sequencediscussed in the above paragraphs may include these modifications.

Also disclosed are isolated polynucleotides that encode the antibodiesor antigen-binding fragments that immunospecifically bind to GPRC5D. Theisolated polynucleotides capable of encoding the variable domainsegments provided herein may be included on the same, or different,vectors to produce antibodies or antigen-binding fragments. An exemplarypolynucleotide sequence that encodes a GPRC5D antibody is shown below:

Heavy Chain Sequence (SEQ ID NO: 96):atggcctgggtctggaccctgctgttcctgatggccgctgcccagagcatccaggcccaggtgcagctggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctacagcttcaccggctacaccatgaactgggtgcggcaggcccccggccagggcctggagtggatgggcctgatcaacccctacaacagcgacaccaactacgcccagaagctgcagggccgggtgaccatgaccaccgacaccagcaccagcaccgcctacatggagctgcggagcctgcggagcgacgacaccgccgtgtactactgcgcccgggtggccctgcgggtggccctggactactggggccagggcaccctggtgaccgtgagcagcgcctccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaaacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga Light Chain Sequence (SEQ ID NO: 90):atgcgggtgctggcccagctgctgggactgctgctgctgtgcttccctggcgccagatgcgacatccagatgacccagagccccagcagcctgagcgccagcgtgggcgaccgggtgaccatcacctgcaaggccagccagaacgtggccacccacgtgggctggtaccagcagaagcccggcaaggcccccaagcggctgatctacagcgccagctaccggtacagcggcgtgcccagccggttcagcggcagcggcagcggcaccgagttcaccctgaccatcagcaacctgcagcccgaggacttcgccacctactactgccagcagtacaaccggtacccctacaccttcggccagggcaccaagctggagatcaagcgtacggtggcctgcaccatctgtcttcatttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagctt caacaggggagagtgttga

Polynucleotides encoding recombinant antigen-binding proteins also arewithin the scope of the disclosure. In some embodiments, thepolynucleotides described (and the peptides they encode) include aleader sequence. Any leader sequence known in the art may be employed.The leader sequence may include, but is not limited to, a restrictionsite or a translation start site.

The GPRC5D-specific antibodies or antigen-binding fragments describedherein include variants having single or multiple amino acidsubstitutions, deletions, or additions that retain the biologicalproperties (e.g., binding affinity or immune effector activity) of thedescribed GPRC5D-specific antibodies or antigen-binding fragments. Inthe context of the present invention the following notations are, unlessotherwise indicated, used to describe a mutation; i) substitution of anamino acid in a given position is written as e.g. S228P which means asubstitution of a Serine in position 228 with a Proline; and ii) forspecific variants the specific three or one letter codes are used,including the codes Xaa and X to indicate any amino acid residue. Thus,the substitution of Serine for Arginine in position 228 is designatedas: S228P, or the substitution of any amino acid residue for Serine inposition 228 is designated as S228P. In case of deletion of Serine inposition 228 it is indicated by S228*. The skilled person may producevariants having single or multiple amino acid substitutions, deletions,or additions.

These variants may include: (a) variants in which one or more amino acidresidues are substituted with conservative or nonconservative aminoacids, (b) variants in which one or more amino acids are added to ordeleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies or antigen-binding fragments described herein may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In other embodiments, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art.

The GPRC5D-specific antibodies or antigen-binding fragments describedherein may embody several antibody isotypes, such as IgM, IgD, IgG, IgAand IgE. In some embodiments the antibody isotype is IgG1, IgG2, IgG3,or IgG4 isotype, preferably IgG1 or IgG4 isotype. Antibody orantigen-binding fragment thereof specificity is largely determined bythe amino acid sequence, and arrangement, of the CDRs. Therefore, theCDRs of one isotype may be transferred to another isotype withoutaltering antigen specificity. Alternatively, techniques have beenestablished to cause hybridomas to switch from producing one antibodyisotype to another (isotype switching) without altering antigenspecificity. Accordingly, such antibody isotypes are within the scope ofthe described antibodies or antigen-binding fragments.

Also provided are vectors comprising the polynucleotides describedherein. The vectors can be expression vectors. Recombinant expressionvectors containing a sequence encoding a polypeptide of interest arethus contemplated as within the scope of this disclosure. The expressionvector may contain one or more additional sequences such as but notlimited to regulatory sequences (e.g., promoter, enhancer), a selectionmarker, and a polyadenylation signal. Vectors for transforming a widevariety of host cells are well known and include, but are not limitedto, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterialartificial chromosomes (BACs), yeast artificial chromosomes (YACs), aswell as other bacterial, yeast and viral vectors.

Recombinant expression vectors within the scope of the descriptioninclude synthetic, genomic, or cDNA-derived nucleic acid fragments thatencode at least one recombinant protein which may be operably linked tosuitable regulatory elements. Such regulatory elements may include atranscriptional promoter, sequences encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. Expression vectors, especially mammalianexpression vectors, may also include one or more nontranscribed elementssuch as an origin of replication, a suitable promoter and enhancerlinked to the gene to be expressed, other 5′ or 3′ flankingnontranscribed sequences, 5′ or 3′ nontranslated sequences (such asnecessary ribosome binding sites), a polyadenylation site, splice donorand acceptor sites, or transcriptional termination sequences. An originof replication that confers the ability to replicate in a host may alsobe incorporated.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. Exemplary vectors may be constructed as described byOkayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody- or antigen-binding fragment-codingsequence is placed under control of a powerful constitutive promoter,such as the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase,beta-actin, human myosin, human hemoglobin, human muscle creatine, andothers. In addition, many viral promoters function constitutively ineukaryotic cells and are suitable for use with the describedembodiments. Such viral promoters include without limitation,Cytomegalovirus (CMV) immediate early promoter, the early and latepromoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, thelong terminal repeats (LTRs) of Maloney leukemia virus, HumanImmunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous SarcomaVirus (RSV), and other retroviruses, and the thymidine kinase promoterof Herpes Simplex Virus. In one embodiment, the GPRC5D-specific antibodyor antigen-binding fragment thereof coding sequence is placed undercontrol of an inducible promoter such as the metallothionein promoter,tetracycline-inducible promoter, doxycycline-inducible promoter,promoters that contain one or more interferon-stimulated responseelements (ISRE) such as protein kinase R 2′,5′-oligoadenylatesynthetases, Mx genes, ADAR1, and the like.

Vectors described herein may contain one or more Internal Ribosome EntrySite(s) (IRES). Inclusion of an IRES sequence into fusion vectors may bebeneficial for enhancing expression of some proteins. In someembodiments the vector system will include one or more polyadenylationsites (e.g., SV40), which may be upstream or downstream of any of theaforementioned nucleic acid sequences. Vector components may becontiguously linked, or arranged in a manner that provides optimalspacing for expressing the gene products (i.e., by the introduction of“spacer” nucleotides between the ORFs), or positioned in another way.Regulatory elements, such as the IRES motif, may also be arranged toprovide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in theart. Selection markers include positive and negative selection markers,for example, antibiotic resistance genes (e.g., neomycin resistancegene, a hygromycin resistance gene, a kanamycin resistance gene, atetracycline resistance gene, a penicillin resistance gene, a puromycinresistance gene, a blasticidin resistance gene), glutamate synthasegenes, HSV-TK, HSV-TK derivatives for ganciclovir selection, orbacterial purine nucleoside phosphorylase gene for 6-methylpurineselection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acidsequence encoding a selection marker or the cloning site may be upstreamor downstream of a nucleic acid sequence encoding a polypeptide ofinterest or cloning site.

The vectors described herein may be used to transform various cells withthe genes encoding the described antibodies or antigen-bindingfragments. For example, the vectors may be used to generateGPRC5D-specific antibody or antigen-binding fragment-producing cells.Thus, another aspect features host cells transformed with vectorscomprising a nucleic acid sequence encoding an antibody orantigen-binding fragment thereof that specifically binds GPRC5D, such asthe antibodies or antigen-binding fragments described and exemplifiedherein.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of carrying out the described methods, in accordance with thevarious embodiments described and exemplified herein. The technique usedshould provide for the stable transfer of the heterologous gene sequenceto the host cell, such that the heterologous gene sequence is heritableand expressible by the cell progeny, and so that the necessarydevelopment and physiological functions of the recipient cells are notdisrupted. Techniques which may be used include but are not limited tochromosome transfer (e.g., cell fusion, chromosome mediated genetransfer, micro cell mediated gene transfer), physical methods (e.g.,transfection, spheroplast fusion, microinjection, electroporation,liposome carrier), viral vector transfer (e.g., recombinant DNA viruses,recombinant RNA viruses) and the like (described in Cline, 29 Pharmac.Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethyleneglycol (PEG)-induced fusion of bacterial protoplasts with mammaliancells may also be used to transform cells.

Cells suitable for use in the expression of the GPRC5D-specificantibodies or antigen-binding fragments described herein are preferablyeukaryotic cells, more preferably cells of plant, rodent, or humanorigin, for example but not limited to NSO, CHO, CHOK1, perC.6, Tk-ts13,BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa,NS1, Sp2/0 myeloma cells, and BHK cell lines, among others. In addition,expression of antibodies may be accomplished using hybridoma cells.Methods for producing hybridomas are well established in the art.

Cells transformed with expression vectors described herein may beselected or screened for recombinant expression of the antibodies orantigen-binding fragments described herein. Recombinant-positive cellsare expanded and screened for subclones exhibiting a desired phenotype,such as high level expression, enhanced growth properties, or theability to yield proteins with desired biochemical characteristics, forexample, due to protein modification or altered post-translationalmodifications. These phenotypes may be due to inherent properties of agiven subclone or to mutation. Mutations may be effected through the useof chemicals, UV-wavelength light, radiation, viruses, insertionalmutagens, inhibition of DNA mismatch repair, or a combination of suchmethods.

Methods of Using GPRC5D-Specific Antibodies for Treatment

Provided herein are GPRC5D-specific antibodies or antigen-bindingfragments thereof for use in therapy. In particular, these antibodies orantigen-binding fragments may be useful in treating cancer, such asGPRC5D-expressing cancer. Accordingly, the invention provides a methodof treating cancer comprising administering an antibody as describedherein, such as GPRC5D-specific antibodies or antigen-binding fragments.For example, the use may be by interfering with GPRC5D-receptorinteractions or where the antibody is conjugated to a toxin, sotargeting the toxin to the GPRC5D-expressing cancer. In some embodimentsGPRC5D-expressing cancer includes lymphoma, such as multiple myeloma(MM). The antibodies for use in these methods include those describedherein above, for example a GPRC5D-specific antibody or antigen-bindingfragment with the features set out in Table 1, for example the CDRs orvariable domain sequences, and in the further discussion of theseantibodies.

In some embodiments described herein, immune effector properties of theGPRC5D-specific antibodies may be enhanced or silenced through Fcmodifications by techniques known to those skilled in the art. Forexample, Fc effector functions such as C1q binding, complement dependentcytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity(ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.may be provided and/or controlled by modifying residues in the Fcresponsible for these activities.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to acell-mediated reaction in which non-specific cytotoxic cells thatexpress Fc receptors (FcRs) (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell.

The ability of monoclonal antibodies to induce ADCC can be enhanced byengineering their oligosaccharide component. Human IgG1 or IgG3 areN-glycosylated at Asn297 with the majority of the glycans in thewell-known biantennary G0, G0F, G1, G1F, G2 or G2F forms. Antibodiesproduced by non-engineered CHO cells typically have a glycan fucosecontent of about at least 85%. The removal of the core fucose from thebiantennary complex-type oligosaccharides attached to the Fc regionsenhances the ADCC of antibodies via improved Fc.gamma.RIIIa bindingwithout altering antigen binding or CDC activity. Such mAbs can beachieved using different methods reported to lead to the successfulexpression of relatively high defucosylated antibodies bearing thebiantennary complex-type of Fc oligosaccharides such as control ofculture osmolality (Konno et al., Cytotechnology 64:249-65, 2012),application of a variant CHO line Lec13 as the host cell line (Shieldset al., J Biol Chem 277:26733-26740, 2002), application of a variant CHOline EB66 as the host cell line (Olivier et al., MAbs; 2(4), 2010; Epubahead of print; PMID:20562582), application of a rat hybridoma cell lineYB2/0 as the host cell line (Shinkawa et al., J Biol Chem 278:3466-3473,2003), introduction of small interfering RNA specifically against the.alpha. 1,6-fucosyltrasferase (FUT8) gene (Mori et al., BiotechnolBioeng 88:901-908, 2004), or coexpression ofβ-1,4-N-acetylglucosaminyltransferase III and golgi α-mannosidase II ora potent alpha-mannosidase I inhibitor, kifunensine (Ferrara et al., JBiol Chem 281:5032-5036, 2006, Ferrara et al., Biotechnol Bioeng93:851-861, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008).

In some embodiments described herein, ADCC elicited by the GPRC5Dantibodies may also be enhanced by certain substitutions in the antibodyFc. Exemplary substitutions are for example substitutions at amino acidpositions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430(residue numbering according to the EU index) as described in U.S. Pat.No. 6,737,056.

Methods of Detecting GPRC5D

Provided herein are methods for detecting GPRC5D in a biological sampleby contacting the sample with an antibody, or antigen-binding fragmentthereof, described herein. As described herein, the sample may bederived from urine, blood, serum, plasma, saliva, ascites, circulatingcells, circulating tumor cells, cells that are not tissue associated(i.e., free cells), tissues (e.g., surgically resected tumor tissue,biopsies, including fine needle aspiration), histological preparations,and the like. In some embodiments the described methods includedetecting GPRC5D in a biological sample by contacting the sample withany of the GPRC5D-specific antibodies or antigen-binding fragmentsthereof described herein.

In some embodiments the sample may be contacted with more than one ofthe GPRC5D-specific antibodies or antigen-binding fragments describedherein. For example, a sample may be contacted with a firstGPRC5D-specific antibody, or antigen-binding fragment thereof, and thencontacted with a second GPRC5D-specific antibody, or antigen-bindingfragment thereof, wherein the first antibody or antigen-binding fragmentand the second antibody or antigen-binding fragment are not the sameantibody or antigen-binding fragment. In some embodiments, the firstantibody, or antigen-binding fragment thereof, may be affixed to asurface, such as a multiwell plate, chip, or similar substrate prior tocontacting the sample. In other embodiments the first antibody, orantigen-binding fragment thereof, may not be affixed, or attached, toanything at all prior to contacting the sample.

The described GPRC5D-specific antibodies and antigen-binding fragmentsmay be detectably labeled. In some embodiments labeled antibodies andantigen-binding fragments may facilitate the detection GPRC5D via themethods described herein. Many such labels are readily known to thoseskilled in the art. For example, suitable labels include, but should notbe considered limited to, radiolabels, fluorescent labels, epitope tags,biotin, chromophore labels, ECL labels, or enzymes. More specifically,the described labels include ruthenium, ¹¹¹In-DOTA,¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase,alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag),acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridinedyes, rhodamine dyes, Alexafluor® dyes, and the like.

The described GPRC5D-specific antibodies and antigen-binding fragmentsmay be used in a variety of assays to detect GPRC5D in a biologicalsample. Some suitable assays include, but should not be consideredlimited to, western blot analysis, radioimmunoassay, surface plasmonresonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis,immunodiffusion, electrochemiluminescence (ECL) immunoassay,immunohistochemistry, fluorescence-activated cell sorting (FACS) orELISA assay.

In some embodiments described herein detection of GPRC5D-expressingcancer cells in a subject may be used to determine that the subject maybe treated with a therapeutic agent directed against GPRC5D.

GPRC5D is present at detectable levels in blood and serum samples. Thus,provided herein are methods for detecting GPRC5D in a sample derivedfrom blood, such as a serum sample, by contacting the sample with anantibody, or antigen-binding fragment thereof, that specifically bindsGPRC5D. The blood sample, or a derivative thereof, may be diluted,fractionated, or otherwise processed to yield a sample upon which thedescribed method may be performed. In some embodiments, GPRC5D may bedetected in a blood sample, or a derivative thereof, by any number ofassays known in the art, such as, but not limited to, western blotanalysis, radioimmunoassay, surface plasmon resonance,immunofluorimetry, immunoprecipitation, equilibrium dialysis,immunodiffusion, electrochemiluminescence (ECL) immunoassay,immunohistochemistry, fluorescence-activated cell sorting (FACS) orELISA assay.

Methods for Diagnosing Cancer

Provided herein are methods for diagnosing GPRC5D-expressing cancer in asubject. In some embodiments GPRC5D-expressing cancer include lymphomas,such as multiple myeloma (MM). In some embodiments, as described above,detecting GPRC5D in a biological sample, such as a blood sample or aserum sample, provides the ability to diagnose cancer in the subjectfrom whom the sample was obtained. Alternatively, in some embodimentsother samples such as a histological sample, a fine needle aspiratesample, resected tumor tissue, circulating cells, circulating tumorcells, and the like, may also be used to assess whether the subject fromwhom the sample was obtained has cancer. In some embodiments, it mayalready be known that the subject from whom the sample was obtained hascancer, but the type of cancer afflicting the subject may not yet havebeen diagnosed or a preliminary diagnosis may be unclear, thus detectingGPRC5D in a biological sample obtained from the subject can allow for,or clarify, diagnosis of the cancer. For example, a subject may be knownto have cancer, but it may not be known, or may be unclear, whether thesubject's cancer is GPRC5D-expressing.

In some embodiments the described methods involve assessing whether asubject is afflicted with GPRC5D-expressing cancer by determining theamount of GPRC5D that is present in a biological sample derived from thesubject; and comparing the observed amount of GPRC5D with the amount ofGPRC5D in a control, or reference, sample, wherein a difference betweenthe amount of GPRC5D in the sample derived from the subject and theamount of GPRC5D in the control, or reference, sample is an indicationthat the subject is afflicted with a GPRC5D-expressing cancer. Inanother embodiment the amount of GPRC5D observed in a biological sampleobtained from a subject may be compared to levels of GPRC5D known to beassociated with certain forms or stages of cancer, to determine the formor stage of the subject's cancer. In some embodiments the amount ofGPRC5D in the sample derived from the subject is assessed by contactingthe sample with an antibody, or an antigen-binding fragment thereof,that immunospecifically binds GPRC5D, such as the GPRC5D-specificantibodies described herein. The sample assessed for the presence ofGPRC5D may be derived from urine, blood, serum, plasma, saliva, ascites,circulating cells, circulating tumor cells, cells that are not tissueassociated (i.e., free cells), tissues (e.g., surgically resected tumortissue, biopsies, including fine needle aspiration), histologicalpreparations, and the like. In some embodiments GPRC5D-expressing cancerincludes hematological cancer, such as multiple myeloma (MM). In someembodiments the subject is a human.

In some embodiments the method of diagnosing a GPRC5D-expressing cancerwill involve: contacting a biological sample of a subject with aGPRC5D-specific antibody, or an antigen-binding fragment thereof (suchas those derivable from the antibodies and fragments provided in Table1), quantifying the amount of GPRC5D present in the sample that is boundby the antibody or antigen-binding fragment thereof, comparing theamount of GPRC5D present in the sample to a known standard or referencesample; and determining whether the subject's GPRC5D levels fall withinthe levels of GPRC5D associated with cancer. In an additionalembodiment, the diagnostic method can be followed with an additionalstep of administering or prescribing a cancer-specific treatment. Inanother embodiment, the diagnostic method can be followed with anadditional step of transmitting the results of the determination tofacilitate treatment of the cancer. In some embodiments thecancer-specific treatment may be directed against GPRC5D-expressingcancers, such as the GPRC5D×CD3 multispecific antibodies describedherein.

In some embodiments the described methods involve assessing whether asubject is afflicted with GPRC5D-expressing cancer by determining theamount of GPRC5D present in a blood or serum sample obtained from thesubject; and comparing the observed amount of GPRC5D with the amount ofGPRC5D in a control, or reference, sample, wherein a difference betweenthe amount of GPRC5D in the sample derived from the subject and theamount of GPRC5D in the control, or reference, sample is an indicationthat the subject is afflicted with a GPRC5D-expressing cancer.

In some embodiments the control, or reference, sample may be derivedfrom a subject that is not afflicted with GPRC5D-expressing cancer. Insome embodiments the control, or reference, sample may be derived from asubject that is afflicted with GPRC5D-expressing cancer. In someembodiments where the control, or reference, sample is derived from asubject that is not afflicted with GPRC5D-expressing cancer, an observedincrease in the amount of GPRC5D present in the test sample, relative tothat observed for the control or reference sample, is an indication thatthe subject being assessed is afflicted with GPRC5D-expressing cancer.In some embodiments where the control sample is derived from a subjectthat is not afflicted with GPRC5D-expressing cancer, an observeddecrease or similarity in the amount of GPRC5D present in the testsample, relative to that observed for the control or reference sample,is an indication that the subject being assessed is not afflicted withGPRC5D-expressing cancer. In some embodiments where the control orreference sample is derived from a subject that is afflicted withGPRC5D-expressing cancer, an observed similarity in the amount of GPRC5Dpresent in the test sample, relative to that observed for the control orreference sample, is an indication that the subject being assessed isafflicted with GPRC5D-expressing cancer. In some embodiments where thecontrol or reference sample is derived from a subject that is afflictedwith GPRC5D-expressing cancer, an observed decrease in the amount ofGPRC5D present in the test sample, relative to that observed for thecontrol or reference sample, is an indication that the subject beingassessed is not afflicted with GPRC5D-expressing cancer.

In some embodiments the amount of GPRC5D in the sample derived from thesubject is assessed by contacting the sample with an antibody, or anantigen-binding fragment thereof, that specifically binds GPRC5D, suchas the antibodies described herein. The sample assessed for the presenceof GPRC5D may be derived from a blood sample, a serum sample,circulating cells, circulating tumor cells, cells that are not tissueassociated (i.e., free cells), tissues (e.g., surgically resected tumortissue, biopsies, including fine needle aspiration), histologicalpreparations, and the like.

In various aspects, the amount of GPRC5D is determined by contacting thesample with an antibody, or antigen-binding fragment thereof, thatspecifically binds GPRC5D. In some embodiments, the sample may becontacted by more than one type of antibody, or antigen-binding fragmentthereof, that specifically binds GPRC5D. In some embodiments, the samplemay be contacted by a first antibody, or antigen-binding fragmentthereof, that specifically binds GPRC5D and then contacted by a secondantibody, or antigen-binding fragment thereof, that specifically bindsGPRC5D. GPRC5D-specific antibodies or antigen-binding fragments such asthose described herein may be used in this capacity.

Various combinations of the GPRC5D-specific antibodies andantigen-binding fragments can be used to provide a “first” and “second”antibody or antigen-binding fragment to carry out the describeddiagnostic methods. In some embodiments GPRC5D-expressing cancerincludes lymphomas, such as multiple myeloma (MM).

In certain embodiments, the amount of GPRC5D is determined by westernblot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation,equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL)immunoassay, immunohistochemistry, fluorescence-activated cell sorting(FACS) or ELISA assay.

In various embodiments of the described diagnostic methods a control orreference sample is used. This sample may be a positive or negativeassay control that ensures the assay used is working properly; forexample, an assay control of this nature might be commonly used forimmunohistochemistry assays. Alternatively, the sample may be astandardized reference for the amount of GPRC5D in a biological samplefrom a healthy subject. In some embodiments, the observed GPRC5D levelsof the tested subject may be compared with GPRC5D levels observed insamples from subjects known to have GPRC5D-expressing cancer. In someembodiments, the control subject may be afflicted with a particularcancer of interest. In some embodiments, the control subject is known tohave early stage cancer, which may or may not be GPRC5D-expressingcancer. In some embodiments, the control subject is known to haveintermediate stage cancer, which may or may not be GPRC5D-expressingcancer. In some embodiments, the control subject is known to have latestage, which may or may not be GPRC5D-expressing cancer.

Methods for Monitoring Cancer

Provided herein are methods for monitoring GPRC5D-expressing cancer in asubject. In some embodiments GPRC5D-expressing cancer includeslymphomas, such as multiple myeloma (MM). In some embodiments thedescribed methods involve assessing whether GPRC5D-expressing cancer isprogressing, regressing, or remaining stable by determining the amountof GPRC5D that is present in a test sample derived from the subject; andcomparing the observed amount of GPRC5D with the amount of GPRC5D in abiological sample obtained, in a similar manner, from the subject at anearlier point in time, wherein a difference between the amount of GPRC5Din the test sample and the earlier sample provides an indication ofwhether the cancer is progressing, regressing, or remaining stable. Inthis regard, a test sample with an increased amount of GPRC5D, relativeto the amount observed for the earlier sample, may indicate progressionof a GPRC5D-expressing cancer. Conversely, a test sample with adecreased amount of GPRC5D, relative to the amount observed for theearlier sample, may indicate regression of a GPRC5D-expressing cancer.

Accordingly, a test sample with an insignificant difference in theamount of GPRC5D, relative to the amount observed for the earliersample, may indicate a state of stable disease for a GPRC5D-expressingcancer. In some embodiments the amount of GPRC5D in a biological samplederived from the subject is assessed by contacting the sample with anantibody, or an antibody fragment thereof, that specifically bindsGPRC5D, such as the antibodies described herein. The sample assessed forthe presence of GPRC5D may be derived from urine, blood, serum, plasma,saliva, ascites, circulating cells, circulating tumor cells, cells thatare not tissue associated (i.e., free cells), tissues (e.g., surgicallyresected tumor tissue, biopsies, including fine needle aspiration),histological preparations, and the like. In some embodiments the subjectis a human.

In some embodiments the methods of monitoring a GPRC5D-expressing cancerwill involve: contacting a biological sample of a subject with aGPRC5D-specific antibody, or antigen-binding fragment thereof (such asthose derivable from the antibodies and fragments provided in Table 1),quantifying the amount of GPRC5D present in the sample, comparing theamount of GPRC5D present in the sample to the amount of GPRC5Ddetermined to be in a biological sample obtained, in a similar manner,from the same subject at an earlier point in time; and determiningwhether the subject's GPRC5D level has changed over time. A test samplewith an increased amount of GPRC5D, relative to the amount observed forthe earlier sample, may indicate progression of cancer. Conversely, atest sample with a decreased amount of GPRC5D, relative to the amountobserved for the earlier sample, may indicate regression of aGPRC5D-expressing cancer. Accordingly, a test sample with aninsignificant difference in the amount of GPRC5D, relative to the amountobserved for the earlier sample, may indicate a state of stable diseasefor a GPRC5D-expressing cancer. In some embodiments, the GPRC5D levelsof the sample may be compared to a known standard or a reference sample,alone or in addition to the GPRC5D levels observed for a sample assessedat an earlier point in time. In an additional embodiment, the diagnosticmethod can be followed with an additional step of administering acancer-specific treatment. In some embodiments the cancer-specifictreatment may be directed against GPRC5D-expressing cancers.

In various aspects, the amount of GPRC5D is determined by contacting thesample with an antibody, or antigen-binding fragment thereof, thatspecifically binds GPRC5D. In some embodiments, the sample may becontacted by more than one type of antibody, or antigen-binding fragmentthereof, that specifically binds GPRC5D. In some embodiments, the samplemay be contacted by a first antibody, or antigen-binding fragmentthereof, that specifically binds GPRC5D and then contacted by a secondantibody, or antigen-binding fragment thereof, that specifically bindsGPRC5D. Antibodies such as those described herein may be used in thiscapacity.

Various combinations of the antibodies and antigen-binding fragmentsdescribed in Table 1 can be used to provide a “first” and “second”antibody or antigen-binding fragment to carry out the describedmonitoring methods. In some embodiments GPRC5D-expressing cancerincludes a hematological cancer, such as multiple myeloma (MM).

In certain embodiments, the amount of GPRC5D is determined by westernblot analysis, radioimmunoassay, immunofluorimetry, immunoprecipitation,equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL)immunoassay, immunohistochemistry, fluorescence-activated cell sorting(FACS) or ELISA assay.

Kits for Detecting GPRC5D

Provided herein are kits for detecting GPRC5D in a biological sample.These kits include one or more of the GPRC5D-specific antibodiesdescribed herein, or an antigen-binding fragment thereof, andinstructions for use of the kit.

The provided GPRC5D-specific antibody, or antigen-binding fragment, maybe in solution; lyophilized; affixed to a substrate, carrier, or plate;or detectably labeled.

The described kits may also include additional components useful forperforming the methods described herein. By way of example, the kits maycomprise means for obtaining a sample from a subject, a control orreference sample, e.g., a sample from a subject having slowlyprogressing cancer and/or a subject not having cancer, one or moresample compartments, and/or instructional material which describesperformance of a method of the invention and tissue specific controls orstandards.

The means for determining the level of GPRC5D can further include, forexample, buffers or other reagents for use in an assay for determiningthe level of GPRC5D. The instructions can be, for example, printedinstructions for performing the assay and/or instructions for evaluatingthe level of expression of GPRC5D.

The described kits may also include means for isolating a sample from asubject. These means can comprise one or more items of equipment orreagents that can be used to obtain a fluid or tissue from a subject.The means for obtaining a sample from a subject may also comprise meansfor isolating blood components, such as serum, from a blood sample.Preferably, the kit is designed for use with a human subject.

Multispecific Antibodies

The binding domains of the anti-GPRC5D antibodies described hereinrecognize cells expressing GPRC5D on their surface. As noted above,GPRC5D expression can be indicative of a cancerous cell. More specifictargeting to particular subsets of cells can be achieved by makingbispecific molecules, such as antibodies or antibody fragments, whichbind to GPRC5D and to another target, such as CD3 and BCMA. This isachieved by making a molecule which comprises a first region binding toGPRC5D and a second binding region binding to the other target antigen.The antigen-binding regions can take any form that allows specificrecognition of the target, for example the binding region may be or mayinclude a heavy chain variable domain, an Fv (combination of a heavychain variable domain and a light chain variable domain), a bindingdomain based on a fibronectin type III domain (such as from fibronectin,or based on a consensus of the type III domains from fibronectin, orfrom tenascin or based on a consensus of the type III domains fromtenascin, such as the Centyrin molecules from Janssen Biotech, Inc., seee.g. WO2010/051274 and WO2010/093627). Accordingly, bispecific moleculescomprising two different antigen-binding regions which bind GPRC5D andanother antigen, respectively, are provided.

Some of the multispecific antibodies described herein comprise twodifferent antigen-binding regions which bind GPRC5D and CD3,respectively. In preferred embodiments, multispecific antibodies thatbind GPRC5D and CD3 (GPRC5D×CD3-multispecific antibodies) andmultispecific antigen-binding fragments thereof are provided. In someembodiments, the GPRC5D×CD3-multispecific antibody comprises a firstheavy chain (HC1) and a first light chain (LC1) that pair to form afirst antigen-binding site that specifically binds GPRC5D and a secondheavy chain (HC2) and a second light chain (LC2) that pair to form asecond antigen-binding site that specifically binds CD3. In preferredembodiments, the GPRC5D×CD3-multispecific antibody is a bispecificantibody comprising a GPRC5D-specific arm comprising a first heavy chain(HC1) and a first light chain (LC1) that pair to form a firstantigen-binding site that specifically binds CD3 and a CD3-specific armcomprising second heavy chain (HC2) and a second light chain (LC2) thatpair to form a second antigen-binding site that specifically bindsGPRC5D. In some embodiments, the bispecific antibodies of the inventioninclude antibodies having a full length antibody structure. “Full lengthantibody” as used herein refers to an antibody having two full lengthantibody heavy chains and two full length antibody light chains.

A full length antibody heavy chain (HC) includes heavy chain variableand constant domains VH, CH1, CH2, and CH3. A full length antibody lightchain (LC) includes light chain variable and constant domains VL and CL.The full length antibody may be lacking the C-terminal lysine (K) ineither one or both heavy chains. The term “Fab-arm” or “half molecule”refers to one heavy chain-light chain pair that specifically binds anantigen. In some embodiments, one of the antigen-binding domains is anon-antibody based binding domain, e.g. a binding domain of based on afibronectin type 3 domain, e.g. Centyrin.

The GPRC5D-binding arm of the multispecific antibodies provided hereinmay be derived from any of the GPRC5D-specific antibodies describedabove. In some exemplary embodiments of such GPRC5D-binding arms, thefirst antigen-binding region which binds GPRC5D comprises a heavy chainCDR1, CDR2, and CDR3 derived from an antibody clone as described inTable 1. In some exemplary embodiments of such GPRC5D-binding arms, thefirst antigen-binding region which binds GPRC5D comprises heavy chainCDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 derived froman antibody clone as described in Table 1. In some exemplary embodimentsof such GPRC5D-binding arms, the first antigen-binding region whichbinds GPRC5D comprises heavy chain CDR1, CDR2, and CDR3 of clone GC5B81,GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385,GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597.

In some exemplary embodiments of such GPRC5D-binding arms, the firstantigen-binding region which binds GPRC5D comprises heavy chain CDR1,CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clone GC5B81,GC5B465, GS5B483 or GC5B596. In some exemplary embodiments of suchGPRC5D-binding arms, the first antigen-binding region which binds GPRC5Dcomprises a heavy chain variable domain derived from an antibody cloneas described in Table 1. In some exemplary embodiments of suchGPRC5D-binding arms, the first antigen-binding region which binds GPRC5Dcomprises heavy chain variable domain and light chain variable domainderived from an antibody clone as described in Table 1. In someexemplary embodiments of such GPRC5D-binding arms, the firstantigen-binding region which binds GPRC5D comprises heavy chain variabledomain of clone GC5B81, GC5B465, GS5B483 or GC5B596. In some exemplaryembodiments of such GPRC5D-binding arms, the first antigen-bindingregion which binds GPRC5D comprises heavy chain variable domain andlight chain variable domain of clone GC5B81, GC5B465, GS5B483, GC5B596,GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603,GC5B599, GC5B601, GC5B598, or GC5B597.

Table 3 provides a listing of GPRC5D×CD3 bi-specific antibodies havingone heavy and light chain pair specific for GPRC5D and another heavy andlight chain pair specific for CD3, where the particular antibody ID islisted to describe the antigen-specific antibody arms used to producethe described embodiment.

TABLE 3 GPRC5D-specific CD3-specific arm = Ab ID arm = Ab ID GC5B81CD3B219 GC5B465 CD3B219 GC5B483 CD3B219 GC5B596 CD3B219 GC5B382 CD3B219GC5B379 CD3B219 GC5B373 CD3B219 GC5B376 CD3B219 GC5B385 CD3B219 GC5B370CD3B219 GC5B602 CD3B219 GC5B603 CD3B219 GC5B599 CD3B219 GC5B601 CD3B219GC5B598 CD3B219 GC5B597 CD3B219

In some embodiments of the bispecific antibodies, the GPRC5D-binding armbinds also binds cynomolgus GPRC5D, preferably the extracellular domainthereof.

In some embodiments, the GPRC5D-binding arm of the multispecificantibody is IgG, or a derivative thereof, e.g., IgG1, IgG2, IgG3, andIgG4 isotypes. In some embodiments wherein the GPRC5D-binding arm has anIgG4 isotype, it contains S228P, L234A, and L235A substitution(s) in itsFc region.

In some embodiments of the bispecific antibodies, the secondantigen-binding arm binds human CD3. In some preferred embodiments, theCD3-specific arm of the GPRC5D×CD3 bispecific antibody is derived from aCD3-specific antibody that binds and activates human primary T cellsand/or cynomolgus monkey primary T cells. In some embodiments, theCD3-binding arm binds to an epitope at the N-terminus of CD3ε. In someembodiments, the CD3-binding arm contacts an epitope including the sixN-terminal amino acids of CD3ε. In some embodiments, the CD3-specificbinding arm of the bispecific antibody is derived from the mousemonoclonal antibody SP34, a mouse IgG3/lambda isotype. In someembodiments, the CD3-binding arm comprises the CDRs of antibody SP34.Such CD3-binding arms may bind to CD3 with an affinity of 5×10⁻⁷M orless, such as 1×10⁻⁷M or less, 5×10⁻⁸M or less, 1×10⁻⁸M or less, 5×10⁻⁹Mor less, or 1×10⁻⁹M or less. The CD3-specific binding arm may be ahumanized version of an arm of mouse monoclonal antibody SP34. Humanframework adaptation (HFA) may be used to humanize the anti-CD3 antibodyfrom which the CD3-specific arm is derived. In some embodiments of thebispecific antibodies, the CD3-binding arm comprises a heavy chain andlight chain pair selected from Table 2.

In some embodiments, the CD3-binding arm is IgG, or a derivativethereof. In some embodiments, the CD3-binding arm is IgG1, IgG2, IgG3,or IgG4. In some embodiments where in the CD3-binding arm has an IgG4isotype, it contains S228P, L234A, L235A, F405L, and R409Ksubstitution(s) in its Fc region. In some embodiments, the antibodies orantigen-binding fragments bind CD3ε on primary human T cells. In someembodiments, the antibodies or antigen-binding fragments bind CD3ε onprimary cynomolgus T cells. In some embodiments, the antibodies orantigen-binding fragments bind CD3ε on primary human and cynomolgus Tcells. In some embodiments, the antibodies or antigen-binding fragmentsactivate primary human CD3+ T cells. In some embodiments, the antibodiesor antigen-binding fragments activate primary cynomolgus CD4+ T cells.

In some embodiments are provided a GPRC5D×CD3 bispecific antibody havinga GPRC5D-binding arm comprising a heavy chain of antibody clone GC5B81,GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385,GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597. Insome embodiments are provided a GPRC5D×CD3 bispecific antibody having aGPRC5D-binding arm comprising a heavy chain and light chain of antibodyclone GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373,GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598,or GC5B597. In some embodiments are provided a GPRC5D×CD3 bispecificantibody having a CD3-binding arm comprising a heavy chain of antibodyclone CD3B219. In some embodiments are provided a GPRC5D×CD3 bispecificantibody having a CD3-binding arm comprising a heavy chain and lightchain of antibody clone CD3B219. In some embodiments are provided aGPRC5D×CD3 bispecific antibody having a GPRC5D-binding arm comprising aheavy chain of antibody clone GC5B81, GC5B465, GS5B483, GC5B596,GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603,GC5B599, GC5B601, GC5B598, or GC5B597 and a CD3-binding arm comprising aheavy chain of antibody clone CD3B219. In some embodiments are provideda GPRC5D×CD3 bispecific antibody having a GPRC5D-binding arm comprisinga heavy chain and light chain of antibody clone GC5B81, GC5B465,GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370,GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597 and aCD3-binding arm comprising a heavy chain and light chain of antibodyclone CD3B219.

An exemplary GPRC5D×CD3 bispecific antibody is provided in Table 23.

Different formats of bispecific antibodies have been described and wererecently reviewed by Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276.

In some embodiments, the bispecific antibody of the present invention isa diabody, a cross-body, or a bispecific antibody obtained via acontrolled Fab arm exchange as those described in the present invention.

In some embodiments, the bispecific antibodies include IgG-likemolecules with complementary CH3 domains to force heterodimerisation;recombinant IgG-like dual targeting molecules, wherein the two sides ofthe molecule each contain the Fab fragment or part of the Fab fragmentof at least two different antibodies; IgG fusion molecules, wherein fulllength IgG antibodies are fused to an extra Fab fragment or parts of Fabfragment; Fc fusion molecules, wherein single chain Fv molecules orstabilized diabodies are fused to heavy-chain constant-domains,Fc-regions or parts thereof; Fab fusion molecules, wherein differentFab-fragments are fused together; ScFv- and diabody-based and heavychain antibodies (e.g., domain antibodies, nanobodies) wherein differentsingle chain Fv molecules or different diabodies or differentheavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused toeach other or to another protein or carrier molecule.

In some embodiments, IgG-like molecules with complementary CH3 domainsmolecules include the Triomab/Quadroma (Trion Pharma/Fresenius Biotech),the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and theelectrostatically-matched (Amgen), the LUZ-Y (Genentech), the StrandExchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonic(Merus) and the DuoBody (Genmab A/S).

In some embodiments, recombinant IgG-like dual targeting moleculesinclude Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody(Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star)and CovX-body (CovX/Pfizer).

In some embodiments, IgG fusion molecules include Dual Variable Domain(DVD)-Ig (Abbott), IgG-like Bispecific (InnClone/Eli Lilly), Ts2Ab(MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb(Roche).

In some embodiments, Fc fusion molecules include to ScFv/Fc Fusions(Academic Institution), SCORPION (Emergent BioSolutions/Trubion,Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART)(MacroGenics) and Dual(ScFv).sub.2-Fab (National Research Center forAntibody Medicine—China).

In some embodiments, Fab fusion bispecific antibodies include F(ab)2(Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL)(ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv(UCB-Celltech). ScFv-, diabody-based and domain antibodies include butare not limited to Bispecific T Cell Engager (BiTE) (Micromet), TandemDiabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART)(MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies(AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) andCOMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dualtargeting heavy chain only domain antibodies.

Full length bispecific antibodies of the invention may be generated forexample using Fab arm exchange (or half molecule exchange) between twomono specific bivalent antibodies by introducing substitutions at theheavy chain CH3 interface in each half molecule to favor heterodimerformation of two antibody half molecules having distinct specificityeither in vitro in cell-free environment or using co-expression. The Fabarm exchange reaction is the result of a disulfide-bond isomerizationreaction and dissociation-association of CH3 domains. The heavy-chaindisulfide bonds in the hinge regions of the parent mono specificantibodies are reduced.

The resulting free cysteines of one of the parent monospecificantibodies form an inter heavy-chain disulfide bond with cysteineresidues of a second parent mono specific antibody molecule andsimultaneously CH3 domains of the parent antibodies release and reformby dissociation-association. The CH3 domains of the Fab arms may beengineered to favor heterodimerization over homodimerization. Theresulting product is a bispecific antibody having two Fab arms or halfmolecules which each bind a distinct epitope, i.e. an epitope on GPRC5Dand an epitope on CD3.

“Homodimerization” as used herein refers to an interaction of two heavychains having identical CH3 amino acid sequences. “Homodimer” as usedherein refers to an antibody having two heavy chains with identical CH3amino acid sequences.

“Heterodimerization” as used herein refers to an interaction of twoheavy chains having non-identical CH3 amino acid sequences.“Heterodimer” as used herein refers to an antibody having two heavychains with non-identical CH3 amino acid sequences.

The “knob-in-hole” strategy (see, e.g., PCT Inti. Publ. No. WO2006/028936) may be used to generate full length bispecific antibodies.Briefly, selected amino acids forming the interface of the CH3 domainsin human IgG can be mutated at positions affecting CH3 domaininteractions to promote heterodimer formation. An amino acid with asmall side chain (hole) is introduced into a heavy chain of an antibodyspecifically binding a first antigen and an amino acid with a large sidechain (knob) is introduced into a heavy chain of an antibodyspecifically binding a second antigen. After co-expression of the twoantibodies, a heterodimer is formed as a result of the preferentialinteraction of the heavy chain with a “hole” with the heavy chain with a“knob”. Exemplary CH3 substitution pairs forming a knob and a hole are(expressed as modified position in the first CH3 domain of the firstheavy chain/modified position in the second CH3 domain of the secondheavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T,T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

Other strategies such as promoting heavy chain heterodimerization usingelectrostatic interactions by substituting positively charged residuesat one CH3 surface and negatively charged residues at a second CH3surface may be used, as described in US Pat. Publ. No. US2010/0015133;US Pat. Publ. No. US2009/0182127; US Pat. Publ. No. US2010/028637 or USPat. Publ. No. US2011/0123532. In other strategies, heterodimerizationmay be promoted by the following substitutions (expressed as modifiedposition in the first CH3 domain of the first heavy chain/modifiedposition in the second CH3 domain of the second heavy chain):L351Y_F405AY407V/T394W, T366I_K392M_T394W/F405A_Y407V,T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,L351Y_Y407A/T366V K409F Y407A/T366A_K409F, or T350V_L351Y_F405AY407V/T350V_T366L_K392L_T394W as described in U.S. Pat. Publ. No.US2012/0149876 or U.S. Pat. Publ. No. US2013/0195849.

In addition to methods described above, bispecific antibodies of theinvention may be generated in vitro in a cell-free environment byintroducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimericantibody from two parent monospecific homodimeric antibodies in reducingconditions to allow disulfide bond isomerization according to methodsdescribed in Inti. Pat. Publ. No. WO2011/131746. In the methods, thefirst monospecific bivalent antibody (e.g., anti-GPRC5D antibody) andthe second monospecific bivalent antibody (e.g., anti-CD3 antibody) areengineered to have certain substitutions at the CH3 domain that promotesheterodimer stability; the antibodies are incubated together underreducing conditions sufficient to allow the cysteines in the hingeregion to undergo disulfide bond isomerization; thereby generating thebispecific antibody by Fab arm exchange. The incubation conditions mayoptimally be restored to non-reducing conditions. Exemplary reducingagents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol(DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably areducing agent selected from the group consisting of:2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl)phosphine. For example, incubation for at least 90 min at a temperatureof at least 20° C. in the presence of at least 25 mM 2-MEA or in thepresence of at least 0.5 mM dithiothreitol at a pH from 5-8, for exampleat pH of 7.0 or at pH of 7.4 may be used.

In addition to the described GPRC5D×CD3-multispecific antibodies, alsoprovided are polynucleotide sequences capable of encoding the describedGPRC5D×CD3-multispecific antibodies. Vectors comprising the describedpolynucleotides are also provided, as are cells expressing theGPRC5D×CD3-multispecific antibodies provided herein. Also described arecells capable of expressing the disclosed vectors. These cells may bemammalian cells (such as 293F cells, CHO cells), insect cells (such asSf7 cells), yeast cells, plant cells, or bacteria cells (such as E.coli). The described antibodies may also be produced by hybridoma cells.

Therapeutic Composition and Methods of Treatment Using MultispecificAntibodies and Multispecific Antigen-Binding Fragments Thereof

The GPRC5D bispecific antibodies discussed above, for example theGPRC5D×CD3 bispecific antibodies discussed above, are useful in therapy.In particular, the GPRC5D bispecific antibodies are useful in treatingcancer. Also provided herein are therapeutic compositions for thetreatment of a hyperproliferative disorder in a mammal which comprises atherapeutically effective amount of a multispecific antibody ormultispecific antigen-binding fragment described herein and apharmaceutically acceptable carrier. In preferred embodiments, themultispecific antibody is a GPRC5D×CD3-multispecific antibody asdescribed herein, or a multispecific antigen-binding fragment thereof,and more preferably a GPRC5D×CD3-bispecific antibody as describedherein, or a GPRC5D×CD3-bispecific antigen-binding fragment thereof. Inone embodiment said pharmaceutical composition is for the treatment of aGPRC5D-expressing cancer, including (but not limited to) the following:GPRC5D-expressing B cell cancers, such as multiple myeloma (MM); andother cancers yet to be determined in which GPRC5D is expressed.Particular bispecific antibodies that may be used to treat cancer, suchas hematological cancer, including the specific cancers discussed above,include antibodies GC5B81, GC5B465, GS5B483 or GC5B596.

The pharmaceutical compositions provided herein comprise: a) aneffective amount of a multispecific antibody or antibody fragment of thepresent invention, and b) a pharmaceutically acceptable carrier, whichmay be inert or physiologically active. In preferred embodiments, themultispecific antibody is a GPRC5D×CD3-multispecific antibody asdescribed herein, or a multispecific antigen-binding fragment thereof,and more preferably a GPRC5D×CD3-bispecific antibody as describedherein, or a GPRC5D×CD3-bispecific antigen-binding fragment thereof. Asused herein, the term “pharmaceutically acceptable carriers” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, and the like that are physiologically compatible.Examples of suitable carriers, diluents and/or excipients include one ormore of water, saline, phosphate buffered saline, dextrose, glycerol,ethanol, and the like, as well as any combination thereof. In manycases, it will be preferable to include isotonic agents, such as sugars,polyalcohols, or sodium chloride in the composition. In particular,relevant examples of suitable carrier include: (1) Dulbecco's phosphatebuffered saline, pH.about.7.4, containing or not containing about 1mg/mL to 25 mg/mL human serum albumin, (2) 0.9% saline (0.9% w/v sodiumchloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain anantioxidant such as tryptamine and a stabilizing agent such as Tween20®.

The compositions herein may also contain a further therapeutic agent, asnecessary for the particular disorder being treated. Preferably, themultispecific antibody or antibody fragment and the supplementary activecompound will have complementary activities that do not adversely affecteach other. In a preferred embodiment, the further therapeutic agent iscytarabine, an anthracycline, histamine dihydrochloride, or interleukin2. In a preferred embodiment, the further therapeutic agent is achemotherapeutic agent.

The compositions of the invention may be in a variety of forms. Theseinclude for example liquid, semi-solid, and solid dosage forms, but thepreferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions. The preferred mode ofadministration is parenteral (e.g. intravenous, intramuscular,intraperitoneal, subcutaneous). In a preferred embodiment, thecompositions of the invention are administered intravenously as a bolusor by continuous infusion over a period of time. In another preferredembodiment, they are injected by intramuscular, subcutaneous,intra-articular, intrasynovial, intratumoral, peritumoral,intralesional, or perilesional routes, to exert local as well assystemic therapeutic effects.

Sterile compositions for parenteral administration can be prepared byincorporating the antibody, antibody fragment or antibody conjugate ofthe present invention in the required amount in the appropriate solvent,followed by sterilization by microfiltration. As solvent or vehicle,there may be used water, saline, phosphate buffered saline, dextrose,glycerol, ethanol, and the like, as well as combination thereof. In manycases, it will be preferable to include isotonic agents, such as sugars,polyalcohol's, or sodium chloride in the composition. These compositionsmay also contain adjuvants, in particular wetting, isotonizing,emulsifying, dispersing and stabilizing agents. Sterile compositions forparenteral administration may also be prepared in the form of sterilesolid compositions which may be dissolved at the time of use in sterilewater or any other injectable sterile medium.

The multispecific antibody or antibody fragment may also be orallyadministered. As solid compositions for oral administration, tablets,pills, powders (gelatin capsules, sachets) or granules may be used. Inthese compositions, the active ingredient according to the invention ismixed with one or more inert diluents, such as starch, cellulose,sucrose, lactose or silica, under an argon stream. These compositionsmay also comprise substances other than diluents, for example one ormore lubricants such as magnesium stearate or talc, a coloring, acoating (sugar-coated tablet) or a glaze.

As liquid compositions for oral administration, there may be usedpharmaceutically acceptable solutions, suspensions, emulsions, syrupsand elixirs containing inert diluents such as water, ethanol, glycerol,vegetable oils or paraffin oil. These compositions may comprisesubstances other than diluents, for example wetting, sweetening,thickening, flavoring or stabilizing products.

The doses depend on the desired effect, the duration of the treatmentand the route of administration used; they are generally between 5 mgand 1000 mg per day orally for an adult with unit doses ranging from 1mg to 250 mg of active substance. In general, the doctor will determinethe appropriate dosage depending on the age, weight and any otherfactors specific to the subject to be treated.

Also provided herein are methods for killing a GPRC5D+ cell byadministering to a patient in need thereof a multispecific antibodywhich binds said GPRC5D and is able to recruit T cells to kill saidGPRC5D+ cell (i.e., T cell redirection). Any of the multispecificantibodies or antibody fragments of the invention may be usedtherapeutically. For example, in one embodiment theGPRC5D×CD3-multispecific antibody may be used therapeutically to treatcancer in a subject.

In a preferred embodiment, multispecific antibodies or antibodyfragments of the invention are used for the treatment of ahyperproliferative disorder in a mammal. In a more preferred embodiment,one of the pharmaceutical compositions disclosed above, and whichcontains a multispecific antibody or antibody fragment of the invention,is used for the treatment of a hyperproliferative disorder in a mammal.In one embodiment, the disorder is a cancer. In particular, the canceris a GPRC5D-expressing cancer, including (but not limited to) thefollowing: GPRC5D-expressing B-cell cancers, such as multiple myeloma(MM); and other cancers yet to be determined in which GPRC5D isexpressed. In preferred embodiments, the multispecific antibody is aGPRC5D×CD3-multispecific antibody as described herein, or amultispecific antigen-binding fragment thereof, and more preferably aGPRC5D×CD3-bispecific antibody as described herein, or aGPRC5D×CD3-bispecific antigen-binding fragment thereof.

Accordingly, the pharmaceutical compositions of the invention are usefulin the treatment or prevention of a variety of cancers, including (butnot limited to) the following: a GPRC5D-expressing cancer, including(but not limited to) the following: GPRC5D-expressing B/Plasma cellcancers, such as acute multiple myeloma (MM) or premalignant myelomassuch as MGUS (Monoclonal Gammopathy of Undetermined Significance) andSMM (Smoldering Multiple myeloma) and plasmacytoma; and other cancersyet to be determined in which GPRC5D is expressed.

Similarly, further provided herein is a method for inhibiting the growthof selected cell populations comprising contacting GPRC5D-expressingtarget cells, or tissue containing such target cells, with an effectiveamount of a multispecific antibody or antibody fragment of the presentinvention, either alone or in combination with other cytotoxic ortherapeutic agents, in the presence of a peripheral blood mononuclearcell (PBMC). In preferred embodiments, the multispecific antibody is aGPRC5D×CD3-multispecific antibody as described herein, or amultispecific antigen-binding fragment thereof, and more preferably aGPRC5D×CD3-bispecific antibody as described herein, or aGPRC5D×CD3-bispecific antigen-binding fragment thereof. In a preferredembodiment, the further therapeutic agent is cytarabine, ananthracycline, histamine dihydrochloride, or interleukin 2. In apreferred embodiment, the further therapeutic agent is achemotherapeutic agent. The method for inhibiting the growth of selectedcell populations can be practiced in vitro, in vivo, or ex vivo.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells; treatments of bone marrow prior to itstransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen. Theconditions of non-clinical in vitro use are readily determined by one ofordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells from bonemarrow prior to autologous transplantation in cancer treatment.Treatment can be carried out as follows. Bone marrow is harvested fromthe patient or other individual and then incubated in medium containingserum to which is added the cytotoxic agent of the invention.Concentrations range from about 10 uM to 1 uM, for about 30 min to about48 hr at about 37° C. The exact conditions of concentration and time ofincubation, i.e., the dose, are readily determined by one of ordinaryskill in the art. After incubation the bone marrow cells are washed withmedium containing serum and returned to the patient by i.v. infusionaccording to known methods. In circumstances where the patient receivesother treatment such as a course of ablative chemotherapy or total-bodyirradiation between the time of harvest of the marrow and reinfusion ofthe treated cells, the treated marrow cells are stored frozen in liquidnitrogen using standard medical equipment.

For clinical in vivo use, a therapeutically effective amount of themultispecific antibody or antigen-binding fragment is administered to asubject in need thereof. For example, the GPRC5D×CD3-multispecificantibodies and multispecific antigen-binding fragments thereof may beuseful in the treatment of a GPRC5D-expressing cancer in a subject inneed thereof. In some embodiments, the GPRC5D-expressing cancer is aB-cell cancer, such as multiple myeloma (MM). In preferred embodiments,the multispecific antibody is a GPRC5D×CD3-multispecific antibody asdescribed herein, or a multispecific antigen-binding fragment thereof,and more preferably a GPRC5D×CD3-bispecific antibody as describedherein, or a GPRC5D×CD3-bispecific antigen-binding fragment thereof. Insome embodiments, the subject is a mammal, preferably a human. In someembodiments, the multispecific antibody or antigen-binding fragment willbe administered as a solution that has been tested for sterility.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation.

Parenteral compositions may be formulated in dosage unit form for easeof administration and uniformity of dosage.

The efficient dosages and the dosage regimens for the multispecificantibodies and fragments depend on the disease or condition to betreated and may be determined by one skilled in the art. An exemplary,non-limiting range for a therapeutically effective amount of a compoundof the present invention is about 0.001-10 mg/kg, such as about 0.001-5mg/kg, for example about 0.001-2 mg/kg, such as about 0.001-1 mg/kg, forinstance about 0.001, about 0.01, about 0.1, about 1 or about 10 mg/kg.

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the multispecific antibody or fragment employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a bispecific antibody of the present invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect.

Administration may e.g. be parenteral, such as intravenous,intramuscular or subcutaneous. In one embodiment, the multispecificantibody or fragment may be administered by infusion in a weekly dosageof calculated by mg/m². Such dosages can, for example, be based on themg/kg dosages provided above according to the following: dose(mg/kg)×70: 1.8. Such administration may be repeated, e.g., 1 to 8times, such as 3 to 5 times. The administration may be performed bycontinuous infusion over a period of from 2 to 24 hr, such as of from 2to 12 hr. In one embodiment, the multispecific antibody or fragment maybe administered by slow continuous infusion over a long period, such asmore than 24 hours, in order to reduce toxic side effects.

In one embodiment, the multispecific antibody or fragment may beadministered in a weekly dosage of calculated as a fixed dose for up toeight times, such as from four to six times when given once a week. Suchregimen may be repeated one or more times as necessary, for example,after six months or twelve months. Such fixed dosages can, for example,be based on the mg/kg dosages provided above, with a body weightestimate of 70 kg. The dosage may be determined or adjusted by measuringthe amount of bispecific antibody of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the GPRC5D antigen bindingregion of the multispecific antibodies of the present invention.

In one embodiment, the multispecific antibody or fragment may beadministered by maintenance therapy, such as, e.g., once a week for aperiod of six months or more.

A multispecific antibody or fragment may also be administeredprophylactically in order to reduce the risk of developing cancer, delaythe onset of the occurrence of an event in cancer progression, and/orreduce the risk of recurrence when a cancer is in remission.

The multispecific antibodies and fragments thereof as described hereinmay also be administered in combination therapy, i.e., combined withother therapeutic agents relevant for the disease or condition to betreated. Accordingly, in one embodiment, the antibody-containingmedicament is for combination with one or more further therapeuticagent, such as a chemotherapeutic agent. In some embodiments, the othertherapeutic agent is cytarabine, an anthracycline, histaminedihydrochloride, or interleukin 2. Such combined administration may besimultaneous, separate or sequential, in any order. For simultaneousadministration the agents may be administered as one composition or asseparate compositions, as appropriate.

In one embodiment, a method for treating a disorder involving cellsexpressing GPRC5D in a subject, which method comprises administration ofa therapeutically effective amount of a multispecific antibody orfragment, such as a GPRC5D×CD3 bispecific antibody described herein, andradiotherapy to a subject in need thereof is provided. In one embodimentis provided a method for treating or preventing cancer, which methodcomprises administration of a therapeutically effective amount of amultispecific antibody or fragment, such as a GPRC5D×CD3 antibodydescribed herein, and radiotherapy to a subject in need thereof.Radiotherapy may comprise radiation or associated administration ofradiopharmaceuticals to a patient is provided. The source of radiationmay be either external or internal to the patient being treated(radiation treatment may, for example, be in the form of external beamradiation therapy (EBRT) or brachytherapy (BT)). Radioactive elementsthat may be used in practicing such methods include, e.g., radium,cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67,technetium-99, iodide-123, iodide-131, and indium-111.

Kits

Also provided herein are includes kits, e.g., comprising a describedmultispecific antibody or antigen-binding fragment thereof andinstructions for the use of the antibody or fragments for killing ofparticular cell types. In preferred embodiments, the multispecificantibody is a GPRC5D×CD3-multispecific antibody as described herein, ora multispecific antigen-binding fragment thereof, and more preferably aGPRC5D×CD3-bispecific antibody as described herein, or aGPRC5D×CD3-bispecific antigen-binding fragment thereof. The instructionsmay include directions for using the multispecific antibody orantigen-binding fragment thereof in vitro, in vivo or ex vivo.

Typically, the kit will have a compartment containing the multispecificantibody or antigen-binding fragment thereof. The multispecific antibodyor antigen-binding fragment thereof may be in a lyophilized form, liquidform, or other form amendable to being included in a kit. The kit mayalso contain additional elements needed to practice the method describedon the instructions in the kit, such a sterilized solution forreconstituting a lyophilized powder, additional agents for combiningwith the multispecific antibody or antigen-binding fragment thereofprior to administering to a patient, and tools that aid in administeringthe multispecific antibody or antigen-binding fragment thereof to apatient.

Diagnostic Uses

The multispecific antibodies and fragments described herein may also beused for diagnostic purposes. Thus, also provided are diagnosticcompositions comprising a multispecific antibody or fragments as definedherein, and to its use. In preferred embodiments, the multispecificantibody is a GPRC5D×CD3-multispecific antibody as described herein, ora multispecific antigen-binding fragment thereof, and more preferably aGPRC5D×CD3-bispecific antibody as described herein, or aGPRC5D×CD3-bispecific antigen-binding fragment thereof. In oneembodiment, the present invention provides a kit for diagnosis of cancercomprising a container comprising a bispecific GPRC5D×CD3 antibody, andone or more reagents for detecting binding of the antibody to GPRC5D.Reagents may include, for example, fluorescent tags, enzymatic tags, orother detectable tags. The reagents may also include secondary ortertiary antibodies or reagents for enzymatic reactions, wherein theenzymatic reactions produce a product that may be visualized. Forexample, the multispecific antibodies described herein, orantigen-binding fragments thereof, may be labeled with a radiolabel, afluorescent label, an epitope tag, biotin, a chromophore label, an ECLlabel, an enzyme, ruthenium, ¹¹¹In-DOTA,¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase,alkaline phosphatase and beta-galactosidase, or poly-histidine orsimilar such labels known in the art.

EMBODIMENTS

The disclosure provided herein also provides the following non-limitingembodiments.1. An isolated antibody, or an antigen-binding fragment thereof, thatbinds specifically to GPRC5D comprising:

a. a heavy chain complementarity determining region 1 (CDR1) having theamino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the aminoacid sequence of SEQ ID NO: 5, and a heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 9;

b. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 2, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 6, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 10;

c. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 3, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 7, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11;

d. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 4, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 8, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12;

e. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 61, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 67, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 72;

f. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 2, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 28, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 30;

g. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 27, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 29, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 73;

h. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 27, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 29, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11;

i. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 62, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 74;

j. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 63, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 69, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 75;

k. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 64, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 70, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 12;

l. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 65, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 68, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 76; or

m. a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 66, aheavy chain CDR2 having the amino acid sequence of SEQ ID NO: 71, and aheavy chain CDR3 having the amino acid sequence of SEQ ID NO: 77.

2. The isolated antibody, or an antigen-binding fragment of embodiment1, wherein

a. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 1, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 5, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 9 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 13, a light chain CDR2having the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 19;

b. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 2, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 6, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 10 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 13, a light chain CDR2having the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 19;

c. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 3, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 7, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 11 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 14, a light chain CDR2having the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 20;

d. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 4, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 8, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 12 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 15, a light chain CDR2having the amino acid sequence of SEQ ID NO: 18, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 21;

e. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 61, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 67, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 72 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 13, a light chain CDR2having the amino acid sequence of SEQ ID NO: 78, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 80;

f. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 2, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 28, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 30 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 13, a light chain CDR2having the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 19;

g. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 27, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 29, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 73 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 14, a light chain CDR2having the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 20;

h. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 27, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 29, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 11 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 14, a light chain CDR2having the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 20;

i. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 62, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 68, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 74 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 14, a light chain CDR2having the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 20;

j. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 63, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 69, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 75 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 13, a light chain CDR2having the amino acid sequence of SEQ ID NO: 78, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 80;

k. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 61, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 67, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 72 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 13, a light chain CDR2having the amino acid sequence of SEQ ID NO: 78, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 80;

l. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 65, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 68, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 76 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 95, a light chain CDR2having the amino acid sequence of SEQ ID NO: 79, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 81; or

m. said antibody comprising said heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 66, said heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 71, and said heavy chain CDR3 having the aminoacid sequence of SEQ ID NO: 77 further comprises a light chain CDR1having the amino acid sequence of SEQ ID NO: 15, a light chain CDR2having the amino acid sequence of SEQ ID NO: 18, and a light chain CDR3having the amino acid sequence of SEQ ID NO: 21.

3. An isolated antibody, or an antigen-binding fragment thereof, thatbinds specifically to GPRC5D, and comprises a variable heavy (VH) chainregion selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55,82, 83, 84, 85, 86, 87, 88, 89, or 91.4. The antibody of embodiment 3, wherein the antibody or antigen bindingfragment thereof comprises a variable light (VL) chain region selectedfrom the group consisting of SEQ ID NOs: 56, 57, 58, 92, 93, or 94.5. The antibody of embodiment 3, wherein the antibody or antigen bindingfragment thereof comprises a VH region selected from the groupconsisting of SEQ ID NOs: 52, 53, 54, 55, 82, 83, 84, 85, 86, 87, 88,89, or 91 and a VL region selected from the group consisting of SEQ IDNOs: 56, 57, 58, 92, 93, or 94.6. The antibody of embodiment 5, wherein the VH chain region comprisesSEQ ID NOs: 52, 53, or 83 paired with a VL chain region comprising SEQID NO: 56.7. The antibody of embodiment 5, wherein the VH chain region comprisesSEQ ID NOs: 54, 84, 85, 86, or 90 paired with a VL chain regioncomprising SEQ ID NO: 57.8. The antibody of embodiment 5, wherein the VH chain region comprisesSEQ ID NOs: 55 or 88 paired with a VL chain region comprising SEQ ID NO:58.9. The antibody of embodiment 5, wherein the VH chain region comprisesSEQ ID NOs: 82 or 87 paired with a VL chain region comprising SEQ ID NO:92.10. The antibody of embodiment 5, wherein the VH chain region comprisesSEQ ID NO: 89 paired with a VL chain region comprising SEQ ID NO: 93.11. The antibody of embodiment 5, wherein the VH chain region comprisesSEQ ID NO: 91 paired with a VL chain region comprising SEQ ID NO: 94.12. The antibody or antigen-binding fragment of any one of embodiments 1to 11 wherein the antibody or antigen-binding fragment thereof binds toa polypeptide having the amino acid sequence of SEQ ID NO: 22.13. The antibody or antigen-binding fragment of any one of embodiments 1to 12 wherein the antibody or antigen-binding fragment is a humanantibody or antigen-binding fragment.14. The antibody or antigen-binding fragment of any one of embodiments 1to 13 wherein the antibody or antigen-binding fragment is recombinant.15. The antigen binding fragment of any one of embodiments 1 to 14wherein the antigen binding fragment is a Fab fragment, a Fab2 fragment,or a single chain antibody.16. The antibody or antigen-binding fragment of any one of embodiments 1to 15 wherein the antibody or antigen-binding fragment thereof are ofIgG1, IgG2, IgG3, or IgG4 isotype.17. The antibody or antigen-binding fragment of any of embodiments 1 to9 is an IgG1 or an IgG4 isotype.18. The antibody of embodiment 17 wherein the IgG1 has a K409Rsubstitution in its Fc region.19. The antibody of embodiment 17 wherein the IgG1 has an F405Lsubstitution in its Fc region.20. The antibody of embodiment 20 wherein the IgG4 has an F405Lsubstitution and an R409K substitution in its Fc region.21. The antibody of embodiment 16 further comprising an S228Psubstitution, an L234A substitution, and an L235A substitution in its Fcregion.22. The antibody or antigen-binding fragment of any one of embodiments 1to 14 wherein the antibody or antigen-binding fragment thereofspecifically binds human GPRC5D and cross reacts to cynomolgus monkeyGPRC5D.23. The antibody or antigen-binding fragment of any one of embodiment 17wherein the antibody or antigen-binding fragment induces ADCC in vitrowith an EC₅₀ of less than about 28 nM.24. An isolated cell expressing the antibody or antigen-binding fragmentof any one of embodiments 1 to 11.25. The cell of embodiment 24 wherein the cell is a hybridoma. 26. Thecell of embodiment 24 wherein the antibody is recombinantly produced.27. An isolated GPRC5D×CD3 bispecific antibody comprising:

a) a first heavy chain (HC1);

b) a second heavy chain (HC2);

c) a first light chain (LC1); and

d) a second light chain (LC2),

wherein the HC1 and the LC1 pair to form a first antigen-binding sitethat specifically binds CD3, and the HC2 and the LC2 pair to form asecond antigen-binding site that specifically binds GPRC5D, or aGPRC5D×CD3-bispecific binding fragment thereof.28. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 27 wherein HC1 comprises SEQ ID NO: 25 and LC1 comprises SEQID NO: 26.29. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 52 and LC2 comprises SEQID NO: 56.30. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 53 and LC2 comprises SEQID NO: 56.31. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 54 and LC2 comprises SEQID NO: 57.32. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 55 and LC2 comprises SEQID NO: 58.33. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 82 and LC2 comprises SEQID NO: 92.34. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 83 and LC2 comprises SEQID NO: 56.35. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 84 and LC2 comprises SEQID NO: 57.36. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 85 and LC2 comprises SEQID NO: 57.37. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 86 and LC2 comprises SEQID NO: 57.38. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 87 and LC2 comprises SEQID NO: 92.39. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 88 and LC2 comprises SEQID NO: 58.40. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 89 and LC2 comprises SEQID NO: 93.41. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 91 and LC2 comprises SEQID NO: 94.42. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 28 wherein HC2 comprises SEQ ID NO: 85 and LC2 comprises SEQID NO: 57.43. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofany one of embodiments 27 to 42 wherein the antibody or bispecificbinding fragment are of IgG1, IgG2, IgG3, or IgG4 isotype.44. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 43 wherein the antibody or bispecific binding fragment isIgG4 isotype.45. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiments 27 to 42 wherein the antibody or bispecific binding fragmentthereof binds GPRC5D on the surface of human myeloma cells.46. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiments 27 to 42 wherein the antibody or bispecific binding fragmentthereof binds GPRC5D on the surface of human multiple myeloma cells.47. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 27 to 42 wherein the antibody or bispecific binding fragmentinduces human T cell activation in vitro with an EC₅₀ of less than about0.22 nM.48. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment ofembodiment 27 to 42 wherein the antibody or bispecific binding fragmentinduces T-cell dependent cytotoxicity of GPRC5D-expressing cells invitro with an EC₅₀ of less than about 0.89 nM.49. An isolated cell expressing the antibody or bispecific bindingfragment of any one of embodiments 27 to 42.50. The cell of embodiment 49 wherein the cell is a hybridoma.51. The cell of embodiment 49 wherein the antibody or bispecific bindingfragment is recombinantly produced.52. A method for treating a subject having cancer, said methodcomprising:

-   -   administering a therapeutically effective amount of the        GPRC5D×CD3 bispecific antibody or bispecific binding fragment of        any one of embodiments 27 to 42 to a patient in need thereof for        a time sufficient to treat the cancer.        53. A method for inhibiting growth or proliferation of cancer        cells, said method comprising:    -   administering a therapeutically effective amount of the        GPRC5D×CD3 bispecific antibody or bispecific binding fragment of        any one of embodiments 27 to 42 to inhibit the growth or        proliferation of cancer cells.        54. A method of redirecting a T cell to a GPRC5D-expressing        cancer cell, said method comprising:    -   administering a therapeutically effective amount of the        GPRC5D×CD3 bispecific antibody or bispecific binding fragment of        any one of embodiments 27 to 42 to redirect a T cell to a        cancer.        55. The method of embodiment 52, 53, or 54 wherein the cancer is        a hematological cancer.        56. The method of embodiment 55 wherein the hematological cancer        is a GPRC5D-expressing B cell cancer.        57. The method of embodiment 56 wherein the GPRC5D-expressing B        cell cancer is multiple myeloma.        58. The method of embodiment 52 comprising administering a        second therapeutic agent.        59. The method of embodiment 58 wherein the second therapeutic        agent is a chemotherapeutic agent or a targeted anti-cancer        therapy.        60. The method of embodiment 59 wherein the chemotherapeutic        agent is cytarabine, an anthracycline, histamine        dihydrochloride, or interleukin 2.        61. The method of embodiment 59 wherein the second therapeutic        agent is administered to said subject simultaneously with,        sequentially, or separately from the bispecific antibody.        62. A pharmaceutical composition comprising the GPRC5D×CD3        bispecific antibody or bispecific binding fragment of any one of        embodiments 27 to 42 and a pharmaceutically acceptable carrier.        63. A method for generating the GPRC5D×CD3 bispecific antibody        or bispecific binding fragment of any one of embodiments 27 to        42 by culturing the cell of any one of embodiments 49 to 51.        64. An isolated synthetic polynucleotide encoding the HC1, the        HC2, the LC1 or the LC2 of the GPRC5D×CD3 bispecific antibody or        bispecific binding fragment of any one of embodiments 27 to 42.        65. A kit comprising the GPRC5D×CD3 bispecific antibody or        bispecific binding fragment as defined in any one of embodiments        27 to 42 and/or a polynucleotide as defined in embodiment 63 and        packaging for the same.

EXAMPLES

The following examples are provided to supplement the prior disclosureand to provide a better understanding of the subject matter describedherein. These examples should not be considered to limit the describedsubject matter. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be apparent to personsskilled in the art and are to be included within, and can be madewithout departing from, the true scope of the invention.

Example 1: Antigens

Due to difficulties in production of recombinant GPRC5D antigens,transfected cell lines displaying GPRC5D [human (SEQ ID NO: 22), cyno(SEQ ID NO: 23), and murine (SEQ ID NO: 24)] were generated usingstandard methods to be used as whole cell antigens for antibodygeneration and characterization studies (Table 4).

TABLE 4 GPRC5D-expressing cell lines Protein Cell Line PromoterResistance Human GPRC5D HEK293T CMV Neomycin Cyno GPRC5D HEK293F CMVBlasticidin

Example 2: Generation of GPRC5D Antibodies Using Phage Display

Two distinct approaches were followed in the generation of GPRC5Dantibodies by phage: standard cell panning (negative selection) and FACScell phage panning (competitive selection).

Standard Cell Phage Panning (Negative Selection):

In-house de novo phage libraries have been described in detail (Shi etal (2010) J. Mol. Biol. 397:385-396; Int. Pat. Publ. No. WO09/085462).These libraries were built on three human VH germline genes (IGHV1-69,3-23, 5-51) and four human VL germline genes (A27, B3, L6, O12) designedto have high diversity in CDR-H3. Three de novo phage libraries(DNP00004-169HC/LC mix, DNP00005-323HC/LC mix and DNP00006-551HC/LC mix)displaying Fab variants on phage coat protein pIX were panned againstGPRC5D-expressing HEK293 G5 stable cells (target cells) for round 1, 3,5 and for rounds 2 and 4 the previous round of amplified Fab-pIX phagewere applied to HEK293 background cells (the negative selection) (seeTable 5). Round 1 de novo Fab-pIX phage bound to the target cells wererecovered for overnight amplification. The round 1 selected phage wasapplied to the background cells for round 2 where the unbound Fab-pIXphage were recovered for overnight negatively selected phageamplification. Another set of positive and negative rounds of panningwere performed, rounds 3 and 4. The final round was performed with thelast round of negatively selected amplified phage split into two panningsamples one for target cells and one for background cell panning.

TABLE 5 Standard cell phage panning flowchart-negative selection PanningRound Cells used as antigen 1 Human GPRC5D HEK293 2 HEK293 3 HumanGPRC5D HEK 293 4 HEK293 5 [split] Human GPRC5D HEK 293 HEK 293

Standard Cell Phage Panning (Background Selection):

The Fab-pIX phage display libraries were added to HEK293 cells followingsimilar environments and incubation times as performed with the standardcell panning procedure previously mentioned (Table 6). After threerounds the DNA corresponding to the HEK293 bound Fab-pIX phage was usedto produce PCR amplicons for NGS. These NGS results would be used in anadditional dynamic subtractive analysis within the NGS2.0 software tohelp in distinguishing possible target specific Fab candidates.

TABLE 6 Standard cell phage panning flowchart-background selectionPanning Round Cells used as antigen 1 HEK293 2 HEK293 3 HEK293

FACS Cell Phage Panning (Competitive Selection)

Three rounds of panning were performed by way of applying Fab-pIX phageto a mixture of target cells and background cells at the same time(Table 7). For rounds 1 and 2 the target cells with Fab-pIX phageattached were sorted by use of the GFP signal. To capture the boundFab-pIX phage from the sorted cells an acid cell lysis was appliedfollowed by an E. coli infection. The round 2 amplified Fab-pIX phageand cell mixture were sorted into two populations in the final round,gated for GFP target cells and gated for non-GFP background cells. Thebound Fab-pIX phage for both cell populations was captured by acid lysisand E. coli infection

TABLE 7 FACS cell phage panning flowchart-competitive selection PanningRound Cells used as antigen 1 Human GPRC5D_GFP HEK293 & HEK 293 2 HumanGPRC5D_GFP HEK293 & HEK 293 3 Human GPRC5D_GFP HEK293 & HEK 293

Next Generation Sequencing (NGS) of Phage Panning

The overnight growth under glucose repression was done for the six ofthe last round panning samples. These cultures were used to makemini-prep DNA, Qiagen QIAspin DNA kit. The six DNA samples were used asPCR template to generate amplicons that measured from HCDR1 to HCDR3.The six amplicons were gel purified and pooled by keeping version 2.1,3.0 separate for the standard cell panning and completely pooled forFACS cell panning. These pooled gel purified amplicons were provided toGenewiz NGS services to process by MiSeq 1×300 technology. Files weredelivered by Genewiz and uploaded into a local server (nas2.0). Withinthis server the NGS2.0 software application was used to load, read, andanalyze the sequence files. The top 88 sequences according to copynumber (>50) and ratio of target cell (+) to background cell (−)sequences (ratio >5:1) were selected for IgG conversion. As onlyvariable heavy chain sequence is determined by NGS, the full heavy chainconstructs had to be electronically constructed into the appropriateframeworks. In addition, since only heavy chain sequence was known eachof the candidates was paired with the four parental light chains (A27,B3, L6, O12). The final conversion for the candidates was done as humanIgG4PAA.

ELISA Screen and Standard Sequencing

From the same DNA prep used for NGS, a restriction enzyme digest andself-ligation was done to excise the gene pIX to enable soluble Fabexpression. Ninety-two colonies, picked for each of the three panningsamples were assessed for Fab expression by ELISA and were sequenced todetermine both heavy chain and light chain by Sanger method. The finalcandidates with diversified sequence within the LCDRs were cloned intomammalian expressions plasmids.

Example 4: Initial Characterization of GPRC5D Antibodies ObtainedThrough Phage Display Technology GPRC5D Binding:

Whole cell phage panning was completed using the human GPRC5D cells asantigen, as described above. Following NGS analysis, only heavy chainsequence was known for each of the candidates. Consequently, each heavychain had to be paired with the 4 parental light chains (A27, B3, L6,O12), resulting in 348 mAbs from 87 Hc sequences identified using NGS.These mAbs were initially evaluated for binding to cyno GPRC5D usingFACS. The cyno GPRC5D cell line was selected for initial screening tomaximize the potential binding signal as the cyno GPRC5D cell line had ahigher expression level than did the human GPRC5D cell line. Briefly,FACS screening was performed by normalizing protein concentration to 1ug/ml and 100 ul of protein was mixed with 200,000 cells per well. ThemAb was allowed to incubate with cells for 1 hour at 4° C. The cellswere then washed three times with PBS and 0.2% FBS. An anti-humansecondary mAb conjugated with PE (Jackson cat #709-116-149) was thenadded as the detection reagent. The cells and secondary antibody wereincubated for 1 hour at 4° C. The cells were then washed three timeswith PBS and 0.2% FBS. The cells were washed again using PBS and 0.2%FBS and then analyzed on FACSarry.

A large number of the hits were observed to bind to cyno GPRC5D,including 15 mAbs with MFI greater than 100,000, 23 mAbs with MFI lessthan 100,000 but greater than 10,000 and 63 mAbs with MFI less than10,000 but greater than 1000. (Table 8).

TABLE 8 FACS binding data for NGS derived mAbs to cyno GPRC5D. Heavychain sequences identified from NGS analysis were paired with eachparental Lc as shown. GC5M29 was used as the control (PH9L3 lightchain). mAbs highlighted have an MFI > 1000 GPRC5D mAb GPRC5D mAb GPRC5DmAb GPRC5D mAb Hc ID [PH9L1 (A27)] MFI [PH9L2 (B3)] MFI [PH9L3 (L6)] MFI[PH9L4 (O12)] MFI GC5H18 GC5B22 198 GC5B23 230 GC5B24 246 GC5B25 31324GC5H17 GC5B66 242 GC5B67 272 GC5B68 2682 GC5B69 17416 GC5H20 GC5B114 199GC5B115 266 GC5B116 253 GC5B117 5037 GC5H15 GC5B158 14407 GC5B159 139766GC5B160 84111 GC5B161 7856 GC5H13 GC5B202 89948 GC5B203 9173 GC5B20415203 GC5MB205 52663 GC5H23 GC5B242 242 GC5B243 116641 GC5B244 249GC5B245 469 GC5H14 GC5B282 487 GC5B283 341 GC5B284 249 GC5B285 87116GC5H22 GC5B326 195 GC5B237 265 GC5B328 248 GC5B329 292 GC5H24 GC5B26 194GC5B27 265 GC5B28 229 GC5B29 590 GC5H19 GC5B70 214 GC5B71 229 GC5B722013 GC5B73 3827 GC5H33 GC5B118 178 GC5B119 222 GC5B120 880 GC5B12143924 GC5H36 GC5B162 86773 GC5B163 21970 GC5B164 122043 GC5B165 1870GC5H31 GC5B206 467 GC5B207 3636 GC5B208 481 GC5B209 1409 GC5H29 GC5B246202 GC5B247 3037 GC5B248 2835 GC5B249 1373 GC5H34 GC5B286 239 GC5B287163990 GC5B288 259 GC5B289 271 GC5H35 GC5B330 202 GC5B331 552 GC5B332191978 GC5B333 1591 GC5H26 GC5B30 204 GC5B31 230 GC5B32 16553 GC5B33 270GC5H27 GC5B74 238 GC5B75 365 GC5B76 829 GC5B77 317 GC5H25 GC5B122 153GC5B123 237 GC5B124 6026 GC5B125 6197 GC5H30 GC5B166 1510 GC5B167 6266GC5B168 32482 GC5B169 327 GC5H47 GC5B210 1730 GC5B211 284 GC5B212 278GC5B213 727 GC5H42 GC5B250 3969 GC5B251 213233 GC5B252 63295 GC5B253 574GC5H37 GC5B290 8839 GC5B291 201024 GC5B292 110940 GC5B293 305 GC5H45GC5B334 160 GC5B335 1434 GC5B336 3228 GC5B337 309 GC5H38 GC5B34 157GC5B35 283 GC5B36 14936 GC5B37 297 GC5H40 GC5B78 162 GC5B79 309 GC5B80428 GC5B81 74140 GC5H43 GC5B126 157 GC5B127 265 GC5B128 268 GC5B129 1283GC5H49 GC5B170 1014 GC5B171 51995 GC5B172 1434 GC5B173 332 GC5H62GC5B214 2565 GC5B215 274 GC5B216 263 GC5B217 1589 GC5H60 GC5B254 161GC5B255 5788 GC5B256 460 GC5B257 308 GC5H94 GC5B294 190 GC5B295 2312GC5B296 745 GC5B298 261 GC5H69 GC5B338 188 GC5B339 260 GC5B340 1798GC5B341 290 GC5H85 GC5B38 10809 GC5B39 1112 GC5B40 110654 GC5B41 4379GC5H96 GC5B82 165 GC5B83 272 GC5B84 1547 GC5B85 3563 GC5H97 GC5B130 174GC5B131 293 GC5B132 306 GC5B133 343 GC5H76 GC5B174 219 GC5B175 3631GC5B176 244 GC5B177 324 GC5H68 GC5B218 21420 GC5B219 310 GC5B220 777GC5B221 762 GC5H79 GC5B256 714 GC5B257 1146 GC5B258 456 GC5B259 1129GC5H71 GC5B298 185 GC5B299 1370 GC5B300 260 GC5B301 292 GC5H93 GC5B342164 GC5B343 247 GC5B344 401 GC5B345 283 GC5H21 GC5B42 219 GC5B43 307GC5B44 2694 GC5B45 1022 GC5H39 GC5B86 192 GC5B87 344 GC5B88 470 GC5B8941027 GC5H50 GC5B134 764 GC5B135 340 GC5B136 600 GC5B137 2087 GC5H28GC5B178 750 GC5B179 6708 GC5B180 1005 GC5B181 535 GC5H53 GC5B222 284GC5B223 264 GC5B224 228 GC5B225 605 GC5H51 GC5B262 159 GC5B263 1013GC5B267 238 GC5B268 309 GC5H64 GC5B302 184 GC5B303 329 GC5B304 240GC5B305 1571 GC5H16 GC5B346 182 GC5B347 259 GC5B348 332 GC5B349 552GC5H65 GC5B46 204 GC5B47 327 GC5B48 1320 GC5B49 392 GC5H32 GC5B90 335GC5B91 340 GC5B92 1848 GC5B93 1780 GC5H54 GC5B138 193 GC5B139 354GC5B140 323 GC5B141 292 GC5H99 GC5B182 196 GC5B183 6421 GC5B184 309GC5B185 280 GC5H52 GC5B226 204 GC5B227 298 GC5B228 247 GC5B229 263GC5H48 GC5B266 268 GC5B267 2160 GC5B268 235 GC5B269 281 GC5H44 GC5B306204 GC5B307 313 GC5B308 302 GC5B309 621 GC5H46 GC5B350 198 GC5B351 287GC5B352 4300 GC5B353 1708 GC5H56 GC5B50 229 GC5B51 36679 GC5B52 386GC5B53 446 GC5H55 GC5B94 255 GC5B95 609 GC5B96 411 GC5B97 350 GC5H98GC5B142 194 GC5B143 407 GC5B144 284 GC5B145 483 GC5H61 GC5B186 543GC5B187 1132 GC5B188 376 GC5B189 333 GC5H92 GC5B320 297 GC5B321 294GC5B322 280 GC5B323 386 GC5H5 GC5B17 243 GC5B18 301 GC5B19 1048 GC5B20384 GC5H59 GC5B98 272 GC5B99 388 GC5B100 661 GC5B101 528 GC5H66 GC5B354223 GC5B355 852 GC5B356 374 GC5B357 500 GC5H80 GC5B54 299 GC5B55 415GC5B56 422 GC5B57 487 GC5H87 GC5B102 383 GC5B103 460 GC5B104 3130GC5B105 456 GC5H67 GC5B146 325 GC5B147 370 GC5B148 383 GC5B149 487GC5H73 GC5B190 236 GC5B191 2556 GC5B192 409 GC5B193 387 GC5H86 GC5B23017885 GC5B231 543 GC5B232 494 GC5B233 620 GC5H70 GC5B270 390 GC5B271 660GC5B272 389 GC5B273 450 GC5H90 GC5B314 219 GC5B315 334 GC5B316 363GC5B317 403 GC5H95 GC5B358 211 GC5B359 344 GC5B360 414 GC5B361 449GC5H74 GC5B58 294 GC5B59 448 GC5B60 477 GC5B61 548 GC5H78 GC5B106 267GC5B107 440 GC5B108 474 GC5B109 497 GC5H77 GC5B150 312 GC5B151 452GC5B152 412 GC5B153 709 GC5H91 GC5B194 296 GC5B195 448 GC5B196 491GC5B197 483 GC5H57 GC5B234 128355 GC5B235 195348 GC5B236 134008 GC5B23711255 GC5H41 GC5B274 313 GC5B275 7431 GC5B278 435 GC5B279 542 GC5H58GC5B318 2608 GC5B319 5791 GC5B320 49616 GC5B321 4823 GC5H63 GC5B362 313GC5B363 392 GC5B364 461 GC5B365 3613 GC5H72 GC5B62 326 GC5B63 463 GC5B64485 GC5B65 694 GC5H75 GC5B110 208487 GC5B111 692 GC5B112 140269 GC5B113104136 GC5H81 GC5B154 351 GC5B155 412 GC5B156 440 GC5B157 555 GC5H82GC5B198 285 GC5B199 593 GC5B200 499 GC5B201 485 GC5H83 GC5B238 3698GC5B239 4370 GC5B240 4676 GC5B241 825 GC5H88 GC5B278 3659 GC5B279 481GC5B280 425 GC5B281 633 GC5H89 GC5B332 299 GC5B333 561 GC5B334 4467GC5B335 494 GC5H84 GC5B366 544 GC5B367 474 GC5B368 6313 GC5B369 26116

The 40 mAbs with the highest binding affinity were selected foradditional characterization, which consisted of repeating the bindingstudy to cyno GPCR5D cells, as well as, assessment of binding to humanGPRC5D expressing cells using FACS (Table 9). These data were analyzedto select 17 mAbs for purification and GPRC5D×CD3 bispecific antibodygeneration (highlighted). Factors used to select mAbs for purificationand GPRC5D×CD3 bispecific antibody generation include specificity ofbinding to human GPRC5D, cross-reactivity to cyno GPRC5D and diversityin Hc sequence. For example, GC5H36 was paired with three distinct lightchains and analyzed for binding (GC5B162, GC5B163, GC5B164). OnlyGC5B164 was advanced as this mAb was observed to have the higher MFI tohuman GPRC5D than GC5B162 or GC5B163.

TABLE 9 FACS binding data for NGS derived mAbs to cyno and human GPRC5Dexpressing HEK293F cells. Non-transfected HEK293F cells were used toevaluate binding specificity for GPRC5D. TF7M1636 was used as theisotype control. mAbs highlighted were selected for further analysis.Protein HC Peptide cyno GPRC5D human GPRC5D 293F ID ID MFI MFI MFIGC5B38 GC5H85 6,864 280 215 GC5B110 GC5H75 142,277 2,543 235 GC5B158GC5H15 4,923 285 235 GC5B162 GC5H36 75,139 2,981 230 GC5B202 GC5H1330,470 2,152 256 GC5B218 GC5H68 10,451 277 263 GC5B230 GC5H86 19,103 364290 GC5B234 GC5H57 134,258 1,657 261 GC5B51 GC5H56 22,661 667 480GC5B159 GC5H15 129,370 1,987 258 GC5B163 GC5H36 7,605 840 310 GC5B171GC5H49 24,133 1,144 1,321 GC5B235 GC5H57 158,011 2,643 254 GC5B243GC5H23 67,848 20,458 221 GC5B251 GC5H42 74,510 11,418 235 GC5B281 GC5H34109,418 5,317 570 GC5B291 GC5H37 77,798 10,427 880 GC5B32 GC5H26 3,59714,656 358 GC5B36 GC5H38 5,353 2,002 352 GC5B40 GC5H85 67,279 4,510 310GC5B112 GC5H75 104,875 470 433 GC5B160 GC5H15 40,881 635 276 GC5B164GC5H36 78,672 12,664 343 GC5B168 GC5H30 41,779 28,885 1,238 GC5B204GC5H13 5,090 390 212 GC5B236 GC5H57 78,546 1,442 192 GC5B252 GC5H4222,010 1,498 204 GC5B292 GC5H37 63,366 1,872 480 GC5B320 GC5H58 63,34522,891 231 GC5B332 GC5H35 106,515 13,348 253 GC5B25 GC5H18 22,079 705431 GC5B69 GC5H17 1,593 851 284 GC5B81 GC5H40 62,756 9,986 288 GC5B89GC5H39 31,613 1,068 251 GC5B113 GC5H75 66,475 550 323 GC5B121 GC5H3335,493 3,061 299 GC5B205 GC5H13 63,610 25,199 260 GC5B237 GC5H57 2,579267 248 GC5B285 GC5H14 81,405 19,870 225 GC5B369 GC5H84 16,241 559 252TF7M1636 956 2,621 1,538 human 2nd 209 316 228 anti hGPRC5D 9,001 379 70mouse anti 60 82 65 isotype unstained 44 50 33

The concentration dependent binding profile of each of the selected mAbsagainst human GPRC5D expressing HEK293 cells and non-transfected HEK293cells was determined using FACS (FIG. 1 ). All mAbs were observed tobind to human GPRC5D in a dose dependent manner. Three mAbs, GC5B36,GC5B168, and GC5B205 were also observed to bind to the non-transfected(GPRC5D null) HEK293 cells and were deprioritized due to thisnon-specific interaction with the cells.

The remaining 14 mAbs were selected for bispecific antibody generationwith an anti-CD3 arm CD3B219 and anti-RSV null arm B23M46 (Table 10).

TABLE 10 The relationship between GPRC5D mAb IDs and bispecific IDs.GPRC5D x CD3 x B23Null mAb Protein bispecific bispecific ID ID IDGC5B320 GCDB44 GCDB30 GC5B243 GCDB40 GCDB26 GC5B285 GCDB43 GCDB29GC5B332 GCDB45 GCDB31 GC5B164 GCDB35 GCDB21 GC5B251 GCDB41 GCDB27 GC5B81GCDB32 GCDB18 GC5B235 GCDB38 GCDB24 GC5B110 GCDB34 GCDB20 GC5B202 GCDB36GCDB22 GC5B234 GCDB37 GCDB23 GC5B252 GCDB42 GCDB28 GC5B236 GCDB39 GCDB25GC5B89 GCDB33 GCDB19

One bispecific antibody, GCDB38, precipitated during recombination andanother, GCDB36, contained >10% aggregate. All other bispecificantibodies passed the standard release criteria and the null armbispecific antibodies were analyzed for concentration dependent bindingto human GPRC5D expressing HEK293 cells (FIG. 2 ).

All bispecific antibodies were observed to bind in a dose dependentmanner. GC5B320 was included as a binding comparator to understandbinding differences between bivalent mAbs and monovalent bispecificantibodies. GCDB44 was included as a binding comparator to understandbinding differences between anti-CD3 bispecific antibodies and anti-RSVnull arm mAbs. The expected decrease in apparent binding affinity wasobserved when comparing the mAb GC5B320 to bispecific antibodies GCDB30and GCDB44. In addition, the GCDB44 (anti-CD3 bispecific Ab) wasobserved to have slightly higher binding affinity to the anti-humanGPRC5D cells compared with the GCDB30 (anti-RSV null arm bispecific Ab),demonstrating that the anti-CD3 arm is positively impacted binding tothis cell line.

The panel of bispecific antibodies was also profiled for binding againstMM1R and H929 cells, which endogenously express GPRC5D (FIG. 3 ). Thebinding profile of the bispecific antibodies on MM1R and H929 cells wascompared with the overexpressed human GPCR5D HEK293 cells andnon-transfected HEK293 cells using FACS. The bispecific antibodies wereobserved to bind to GPRC5D as expressed endogenously on MM1R and H929cells with a range of affinity. The highest binding was observed to thehuman GPRC5D HEK cells, which as an overexpressed stable cell line, hasa much higher receptor density than either MM1R or H929 cells. GCDB37,GCDB38, GCDB39 and GCDB41 were not advanced due to the low bindingaffinity observed for H929 and MM1R cells.

In vitro T-Cell Dependent Cytotoxicity

The panel of bispecific antibodies were then profiled for potency in aT-cell mediated cytotoxicity assay using H929 and MM1R target cells(FIGS. 4A and B, Table 11). Briefly, target cells (H929, MM1.R, OPM2,LP-1 and Daudi or HEK parent and HEK+GPRC5D cells) were counted and 10million cells were centrifuged at 1350 rpm for 3 minutes and cellpellets were resuspended in 1 ml of diluted CFSE solution (CellTraceCFSE proliferation stain was reconstituted in 18 μl of sterile DMSO and1 μl of the solution was diluted in 10 ml of sterile PBS) and incubatedat room temperature for 8 minutes in dark. After the incubation, 1 ml ofHI FBS was added to cell suspension to quench the surplus CFSE. Cellswere washed twice in RPMI-1640 with 10% FBS. After reconstitution in 10ml of RPMI, cells were counted and cell viability was recoded in aspreadsheet. Cells were diluted to 2.2×10{circumflex over ( )}5/ml andincubated at 37° C. until use.

Pan T cells from normal donors were thawed in 37° C. water bath, andcells were then centrifuged at 1350 rpm at 4° C. for 3 minutes. Thesupernatants were discarded and reconstituted in culture medium at1.1×10{circumflex over ( )}6/ml concentration. 2×10{circumflex over( )}5 target cells were added to wells of a 96-well U-bottom plate,followed by Fc blocker (to final concentration of 2 mg/ml). All celllines were incubated at room temperature for 10 minutes to block Fcreceptor activity. 1×10{circumflex over ( )}5 T cells were added to thewells (5:1 Effector:Target ratio). After target and T cells were mixed,20 μl of GPRC5D×CD3 bispecific Ab dilutions were added to each well.GPRC5D×CD3 bispecific antibodies were diluted to 800 μg/ml (10×) in PBS.The titration was prepared in 4-fold serial dilutions in PBS in a96-well U-bottom plate. The last column was left as PBS alone (vehiclecontrol). The plates were incubated at 37° C. with 5% CO₂ for 48 hours.

Two days later (48 hours), the plates were centrifuged and 100 μl ofsupernatants were stored at −80° C. for cytokine release assay. Cellswere washed in 200 μl of PBS and incubated in 50 μl of near-IR Live/Deadstain (1:200 dilution) and anti-CD25 PE antibody (1:50 dilution) for 20minutes at room temperature. Then, the cells were washed once in 200 μlof FACS buffer and finally reconstituted in 150 μl of FACS buffer. Cellswere analyzed using FACSCanto II and FlowJo 7.6 for target cytotoxicity(% target) and T cell activation CD25+(% live T cells). Graphing andfitting of data were done in GraphPad Prism 6 using nonlinear regressionwith variable slope (four parameters) function using least squaresmethod.

TABLE 11 Average EC₅₀ calculated from T-cell mediated cytotoxicityassessment of GPRC5D × CD3 bispecific antibodies using H929 and MM1Rtarget cells. GPRC5D × CD3 EC50 (nM) Bispecific Ranking (Average) IDH929 (n = 5) MM1R (n = 3) H929/MM1R GCDB32 0.39 ± 0.3  0.12 ± 0.1  2/1GCDB33 4.29 ± 1.28  0.8 ± 0.18 7/6 GCDB34 2.13 ± 0.93 0.77 ± 0.24 5/5GCDB35 1.39 ± 0.84 0.74 ± 0.32 4/4 GCDB36  5.2 ± 0.93 4.22 ± 0.55 8/9GCDB40  1.2 ± 0.92 0.52 ± 0.38 3/3 GCDB41 NA 2.58 ± 0.15 10/8  GCDB430.36 ± 0.41 0.21 ± 0.29 1/2 GCDB44 10.29 ± 2.65  4.41 ± 0.29  9/10GCDB45 2.25 ± 1   0.91 ± 0.93 6/7

All bispecific antibodies are active in T-cell mediated H929 cellkilling, with a range of potencies observed (Table 11). Similar rankorder was observed for both cell lines. However a lower EC₅₀ wasobserved in MM1R cells. Interestingly, binding affinity was notnecessarily correlative of potency in T-cell mediated cytotoxicityassay. For example, GCDB44 was the highest affinity binding bispecificantibody but the least potent in the cytotoxicity assay. While GCDB43,with similar, albeit slightly lower, binding affinity was the mostpotent in the cytotoxicity assay.

To assess the functional cross-reactivity with cyno GPRC5D, the panel ofbispecific antibodies was then profiled for T-cell mediated cytotoxicityand T cell activation using cyno GPRC5D HEK293 cells (FIGS. 5A and B).All bispecific antibodies were active in this assay though a range ofpotency was observed against the cyno GPRC5D+ expressing cells.

In Vivo Efficacy

A benchmarking in vivo study was then completed to understand the invivo potency of these GPRC5D×CD3 bispecific antibodies. GCDB32 andGCDB35 (FIGS. 5A & B) were selected to test in a H929 prophylactic tumormodel. H929 cells were implanted into NSG mice, one week followinginjection of human PBMCs. Treatment of the bispecific antibodies wasinitiated at the same time that the H929 cells were implanted andcontinued every 2 or 3 days (q2d or q3d) at 10 ug, 1 ug, and 0.1ug/animal doses for a total of five treatments. Ten mice were used ineach group and PBS included as the vehicle control. Treatment wasstopped at day 11 and the study was terminated on day 25 (FIGS. 6A andB) or day 26 (FIGS. 12A-D). All the GPRC5D×CD3 antibodies tested in thisprophylactic model showed 100% tumor growth inhibition at the 10 and 1ug/animal dose except for GCDB35 which showed round 80% tumor growthinhibition at 1 ug/animal dose. At tenfold lower dose (0.1 ug/animal)these bispecific antibodies showed varying degree of efficacy rangingfrom 10 to 80% tumor growth inhibition.

In Vitro T-Cell Dependent Cytotoxicity in Presence of Fc Blocker

To gain an understanding of the specificity of the GPRC5D targeting arm,the panel of GPRC5D×CD3 bispecific antibodies was then assessed in aT-cell redirection cytotoxicity assay in the presence of Fc block. Thisexperiment was critical to understanding specificity as the target cellsfor bispecific antibodies are B-cells expressing Fcγ receptorscapability of interacting with the Fc portion of the bispecific antibodyin the in vitro assay. A shift in potency was observed in the T-cellmediated cytotoxicity assay for a number of the bispecific antibodies,with the greatest shift observed for GCDB40 and GCDB34 (FIG. 7A-7B).

Direct measurement of binding interactions between the four most potentbispecific antibodies (GCDB32, GCDB35, GCDB40, and GCDB43) and Fcγreceptors was then completed (FIG. 8A-8D). One round of Alpha Screenanalysis was carried out for each of the Fcγ receptors and bispecificantibodies listed above. All samples were assayed in duplicate. OnFcγRI, the four bispecific antibodies behave like B21M hIgG4 PAA. Thatis, they are no more competitive than matched isotype control. Similardifferences were also observed between the hIgG1 WT control and the fourbispecific antibodies on FcγRIIIa, with GCD43 most like the IgG4PAAisotype control and the other bispecific antibodies having slightlyhigher affinity for FcγRIIIa. On both FcγRIIa and FcγRIIb, the fourbispecific antibodies compete in the following order:GCDB40>GCDB32>GCDB43>GCDB35. GCDB40 is the most competitive or bindswith highest affinity to FcγRIIa and FcγRIIb. In fact GCDB40 competes onFcγRIIa and FcγRIIb to the same extent as hIgG1 WT, corroborating theobserved shift in potency observed in the T-cell mediated cytotoxicityassay when Fc block was included. Due to the unexpected interactionswith FcγRIIa and FcγRIIb, GCDB40 was not advanced.

Competition Binning Assay

Anti-GPRC5D mAbs were assessed for competition binding with each otherto the human GPCR5D cells. Briefly, cells were plated at 50,000cells/well in 50 uL of media and allowed to settle for 90 minutes at 37C. Wells were then blocked using 3% BSA for 1 hour at room temperature.mAbs were labeled with ruthenium (II) tris-bipyridine,N-hydroxysuccinimide (Ru-label) following standard procedures. In aseparate 96 well plate, 5 uM of competitor mAb was incubated with 50 nMof Ru-labeled mAb. The blocking solution was removed from the cell plateand 25 uL of mAb solution added. Plates were incubated for 1 hour atroom temperature with shaking. After washing the plates thrice with PBS,150 uL of MSD read buffer (without surfactant) was added and the bindingof the Ru-labeled antibody detected using a MSD plate reader.

All mAbs belong to the same competition group, with only GC5B420 andGC5B421 not fully competed by GC5B81, GC5B285, and/or GC5B332 (Blocked<70%) (Table 12). It is hypothesized that simultaneous binding of twomAbs to GPRC5D may be sterically impossible given the small size of theextracellular domain of GPRC5D compared to the size of a mAb.

TABLE 12 Competition binding epitope binning of anti-GPRC5D mAbs.Anti-GPRC5D mAbs were assessed for competition binding with each otherto the human GPCR5D cells (+/− = Blocked < 70%). mAb ID GC5B81 GC5B164GC5B285 GC5B332 GC5B420 GC5B421 GC5B81 + + + + +/− +/−GC5B164 + + + + + + GC5B285 + + + + + +/− GC5B332 + + + + +/− +GC5B420 + + + + + + GC5B421 + + + + + + GC5B243 + + + + + +

Example 4: Generation of GPRC5D Antibodies Using Hydridoma Technology

Three Balb/c mice were immunized intradermally-base of tail withpCMV6-neo (CMV promoter) plasmid DNA expressing full length human GPRC5Don days 0, 10, and 20. Mice received final intraperitoneal andintravenous immunizations on day 59 with Rat Basophilic Leukemia (RBL)cells over-expressing full length human GPRC5D. On day 63, lymph nodesand spleens were harvested, enrichment of B cells was performed, and thecells were used to generate 3500 mAb secreting hybridomas.

Example 5: Initial Characterization of GPRC5D Antibodies ObtainedThrough Hyrbridoma Technology GPRC5D Binding

The hybridomas were screened using FACS for binding to both RBL andhuman GPRC5D expressing RBL cells. The ratio of background adjusted MFIof each mAb binding to the GPRC5D_RBL cells relative to thenon-transfected RBL cells was calculated and any sample with a bindingratio greater than 3 were considered potentially positive. Ninety ninehybridomas had a ratio greater than 3 and were advanced for v-regioncloning. Thirty one mAbs sequences were identified, synthesized,expressed and purified. Two of the 31 mAbs exhibited binding toRBL_GPRC5D cells above background (FIGS. 9A and 8B). GCDB390 and GCDB396were selected for expression, purification and bispecific antibodygeneration with anti-CD3 arm CD3B219 to generate the bispecificantibodies GCDB46 and GCDB47, respectively.

T-Cell Dependent Cytotoxicity GCDB46 and GCDB47 were assessed in theT-cell mediated cytotoxicity assay (FIGS. 10A and 10B). Both bispecificantibodies were observed to be potent, with reported EC₅₀ of 0.67 and0.1 nM respectively. On the basis of these data both mAbs were advancedto lead optimization along with the three phage derived mAbs.

Example 6: Hit Evaluation, Selection and Optimization

Five GPRC5D bispecific antibodies were selected for lead optimization(GCDB32, GCDB43, GCDB35, GCDB46, GCDB47) based on binding, function,cross-reactivity and selectivity data summarized in Table 13.

TABLE 13 Lead optimization data for GPRC5D × CD3 bispecific antibodies.Bispecific antibodies were assessed for binding, function,cross-reactivity and selectivity. Selectivity T cell mediated Targetcell killing GPRC5D × Binding to H929 EC50 ± SD MM1R EC50 ± H929 No Fcvs Cyno GPRC5D CD3 ID GPRC5A, B, C (nM) SD (nM) Fc Block EC50 (nM)GCDB32 No Binding 0.43 ± 0.29 0.12 ± 0.01 2.4 0.19 GCDB43 No Binding0.39 ± 0.07 0.54 2.8 0.95 GCDB40 No Binding  1.2 ± 0.83 0.52 ± 0.31 5.110.61 GCDB35 Low 1.39 ± 0.75 0.74 ± 0.26 2.7 4.65 GCDB34 No Binding 2.01± 0.74 0.77 ± 0.09 6.3 1.14 GCDB45 No Binding 2.36 ± 0.84 0.91 ± 0.762.2 2.82 GCDB33 No Binding 5.21 ± 1.21  0.8 ± 0.15 1.5 0.2 GCDB36 Low5.57 ± 0.47 4.22 ± 0.29 3.35 GCDB46 No Binding 0.67 GCDB47 No Binding0.1 GCDB48 No Binding 0.17 Not Active

Lead optimization was aimed at addressing potential post-translationmodification (PTM) sequence risks for GCDB32 (GC5B81 parent mAb), GCDB43(GC5B285 parent mAb) and GCDB35 (GC5B164 parent mAb) as outlined inTable 14.

TABLE 14PTM mitigation sequence potential liabilities for Phage derived hits. The Hc CDR sequences are shown with potential PTM sequence liabilities are underlined. (SEQ ID NOs for each listed sequence are provided in parenthesis) GPCR5D GPRC5D ID Hc ID Hc CDR1 Hc CDR2 HcCDR3GC5B81 GC5H40 SYAIS (1)  GIIPIFGTANYAQKFQG ESRWRGYKLD  (5) (9) GC5B285GC5H14 NYWMS (2) GISYSGGSKYYADSVKG AAWDFGRRAVRLDY (28) (30) GC5B164GC5H36 SYWIG (27) IIYPGDSDTRYSPSFQG VYSFGGRHKALFDY  (29) (11)

All of the PTM variants assayed for GC5B81 had a substantial reductionin binding affinity, demonstrating the criticality of this residue (HCW102) to the paratope (Table 15).

TABLE 15CDR sequences and binding data of the GC5B81 PTM library. The site of mutation isunderlined. Binding was classified as MFI >10,000 = ++; MFI >1,000 = +; MFI <1,000(SEQ ID NOs for each listed sequence are provided in parenthesis) Heavy Human GPRC5D  Chain  GPRC5D ID ID Mutation Hc CDR1 Hc CDR2 Hc CDR3(FACS) GC5B427 GC5H199 W102Y SYAIS (1) GIIPIFGTANYAQKFQG ESRYRGYKLDY −(5) (31) GC5B428 GC5H198 W102V SYAIS (1) GIIPIFGTANYAQKFQG ESRVRGYKLDY −(5) (32) GC5B430 GC5H196 W102G SYAIS (1) GIIPIFGTANYAQKFQG ESRGRGYKLDY −(5) (33) GC5B431 GC5H195 W102A SYAIS (1) GIIPIFGTANYAQKFQG ESRARGYKLDY −(5) (34) GC5B429 GC5H197 W102F SYAIS (1) GIIPIFGTANYAQKFQG ESRFRGYKLDY −(5) (35)

Binding studies identified a number of mutations for both GC5B285 andGC5B164 which retained binding to human GPRC5D (Tables 16 and 17).

TABLE 16CDR sequences and binding data of the GC5B164 PTM library. Site of mutation isunderlined. Binding was classified as MFI >10,000 = ++; MFI >1,000 = +; MFI <1,000.(SEQ ID NOs for each listed sequence are provided in parenthesis) HumanGPR5D GPRC5D Heavy Chain Binding ID ID Mutation Hc CDR1 Hc CDR2 Hc CDR3(FACS) GC5B471 GC5H278 D55A, W33Y SYYIG IIYPGASDTRYSPSFQG VYSFGGRHKALFDY++ (36) (40) (11) GC5B472 GC5H277 D55A, W33V SYVIG IIYPGASDTRYSPSFQGVYSFGGRHKALFDY + (37) (40) (11) GC5B473 GC5H276 D55A, W33F SYFIGIIYPGASDTRYSPSFQG VYSFGGRHKALFDY ++ (3) (40) (11) GC5B474 GC5H275D55A, W33G SYGIG IIYPGASDTRYSPSFQG VYSFGGRHKALFDY + (38) (40) (11)GC5B475 GC5H274 D55A, W33A SYAIG IIYPGASDTRYSPSFQG VYSFGGRHKALFDY + (39)(40) (11) GC5B476 GC5H273 D55S, W33Y SYYIG IIYPGSSDTRYSPSFQGVYSFGGRHKALFDY ++ (36) (41) (11) GC5B477 GC5H272 D55S, W33V SYVIGIIYPGSSDTRYSPSFQG VYSFGGRHKALFDY + (37) (41) (11) GC5B478 GC5H271D55S, W33F SYFIG IIYPGSSDTRYSPSFQG VYSFGGRHKALFDY ++ (3) (41) (11)GC5B479 GC5H270 D55S, W33G SYGIG IIYPGSSDTRYSPSFQG VYSFGGRHKALFDY + (38)(41) (11) GC5B480 GC5H269 D55S, W33A SYAIG IIYPGSSDTRYSPSFQGVYSFGGRHKALFDY + (39) (41) (11) GC5B481 GC5H268 D55K, W33Y SYYIGIIYPGKSDTRYSPSFQG VYSFGGRHKALFDY ++ (36) (7) (11) GC5B482 GC5H267D55K, W33V SYVIG IIYPGKSDTRYSPSFQG VYSFGGRHKALFDY ++ (37) (7) (11)GC5B483 GC5H266 D55K, W33F SYFIG IIYPGKSDTRYSPSFQG VYSFGGRHKALFDY ++ (3)(7) (11) GC5B484 GC5H265 D55K, W33G SYGIG IIYPGKSDTRYSPSFQGVYSFGGRHKALFDY + (38) (7) (11) GC5B485 GC5H264 D55K, W33A SYAIGIIYPGKSDTRYSPSFQG VYSFGGRHKALFDY + (39) (7) (11) GC5B486 GC5H263D55E, W33Y SYYIG IIYPGESDTRYSPSFQG VYSFGGRHKALFDY ++ (36) (42) (11)GC5B487 GC5H262 D55E, W33V SYVIG IIYPGESDTRYSPSFQG VYSFGGRHKALFDY + (37)(42) (11) GC5B488 GC5H261 D55E, W33F SYFIG IIYPGESDTRYSPSFQGVYSFGGRHKALFDY ++ (3) (42) (11) GC5B489 GC5H260 D55E, W33G SYGIGIIYPGESDTRYSPSFQG VYSFGGRHKALFDY − (38) (42) (11) GC5B490 GC5H259D55E, W33A SYAIG IIYPGESDTRYSPSFQG VYSFGGRHKALFDY + (39) (42) (11)GC5B491 GC5H258 D55Y, W33Y SYYIG IIYPGYSDTRYSPSFQG VYSFGGRHKALFDY ++(36) (43) (11) GC5B492 GC5H257 D55Y, W33V SYVIG IIYPGYSDTRYSPSFQGVYSFGGRHKALFDY ++ (37) (43) (11) GC5B493 GC5H256 D55Y, W33F SYFIGIIYPGYSDTRYSPSFQG VYSFGGRHKALFDY ++ (3) (43) (11) GC5B494 GC5H255D55Y, W33G SYGIG IIYPGYSDTRYSPSFQG VYSFGGRHKALFDY + (38) (43) (11)GC5B495 GC5H254 D55Y, W33A SYAIG IIYPGYSDTRYSPSFQG VYSFGGRHKALFDY + (39)(43) (11) GC5B496 GC5H253 W33Y SYYIG IIYPGDSDTRYSPSFQG VYSFGGRHKALFDY ++(36) (29) (11) GC5B497 GC5H252 W33V SYVIG IIYPGDSDTRYSPSFQGVYSFGGRHKALFDY ++ (37) (29) (11) GC5B498 GC5H251 W33F SYFIGIIYPGDSDTRYSPSFQG VYSFGGRHKALFDY ++ (3) (29) (11) GC5B499 GC5H250 W33GSYGIG IIYPGDSDTRYSPSFQG VYSFGGRHKALFDY − (38) (29) (11) GC5B500 GC5H249W33A SYAIG IIYPGDSDTRYSPSFQG VYSFGGRHKALFDY − (39) (29) (11) GC5B501GC5H248 D55A SYWIG IIYPGASDTRYSPSFQG VYSFGGRHKALFDY ++ (27) (40) (11)GC5B502 GC5H247 D55S SYWIG IIYPGSSDTRYSPSFQG VYSFGGRHKALFDY ++ (27) (41)(11) GC5B503 GC5H246 D55K SYWIG IIYPGKSDTRYSPSFQG VYSFGGRHKALFDY ++ (27)(7) (11) GC5B504 GC5H245 D55E SYWIG IIYPGESDTRYSPSFQG VYSFGGRHKALFDY ++(27) (42) (11) GC5B505 GC5H244 D55Y SYWIG IIYPGYSDTRYSPSFQGVYSFGGRHKALFDY ++ (27) (43) (11)

TABLE 17CDR sequences and binding data of the GC5B285 PTM library. Site of mutation isunderlined. Binding was classified as MFI >10,000 = ++; MFI >1,000 = +; MFI<1,000. (SEQ ID NOs for each listed sequence are provided in parenthesis)Human GPRC5D Heavy Chain Hc GPRC5D ID ID Mutation CDR1 Hc CDR2 Hc CDR3(FACS) GC5B463 GC5H234 D62A, W101Y NYWMS GISYSGGSKYYAASVKGAAYDFGRRAVRLDY ++ (2) (44) (48) GC5B432 GC5H228 D62S, W101V NYWMSGISYSGGSKYYASSVKG AAVDFGRRAVRLDY ++ (2) (6) (49) GC5B465 GC5H227D62S, W101F NYWMS GISYSGGSKYYASSVKG AAFDFGRRAVRLDY ++ (2) (6) (97)GC5B433 GC5H223 D62K, W101V NYWMS GISYSGGSKYYAKSVKG AAVDFGRRAVRLDY ++(2) (45) (49) GC5B434 GC5H222 D62K, W101F NYWMS GISYSGGSKYYAKSVKGAAFDFGRRAVRLDY ++ (2) (45) (97) GC5B435 GC5H219 D62E, W101Y NYWMSGISYSGGSKYYAESVKG AAYDFGRRAVRLDY + (2) (46) (48) GC5B436 GC5H217D62E, W101F NYWMS GISYSGGSKYYAESVKG AAFDFGRRAVRLDY + (2) (46) (97)GC5B461 GC5H216 D62E, W101G NYWMS GISYSGGSKYYAESVKG AAGDFGRRAVRLDY − (2)(46) (50) GC5B462 GC5H215 D62E, W101A NYWMS GISYSGGSKYYAESVKGAAADFGRRAVRLDY ++ (2) (46) (51) GC5B437 GC5H214 D62Y, W101Y NYWMSGISYSGGSKYYAYSVKG AAYDFGRRAVRLDY + (2) (47) (48) GC5B438 GC5H213D62Y, W101V NYWMS GISYSGGSKYYAYSVKG AAVDFGRRAVRLDY + (2) (47) (49)GC5B439 GC5H212 D62Y, W101F NYWMS GISYSGGSKYYAYSVKG AAFDFGRRAVRLDY + (2)(47) (97) GC5B440 GC5H211 D62Y, W101G NYWMS GISYSGGSKYYAYSVKGAAGDFGRRAVRLDY − (2) (47) (50) GC5B441 GC5H210 D62Y, W101A NYWMSGISYSGGSKYYAYSVKG AAADFGRRAVRLDY + (2) (47) (51) GC5B442 GC5H209 W101YNYWMS GISYSGGSKYYADSVKG AAYDFGRRAVRLDY + (2) (28) (48) GC5B443 GC5H208W101V NYWMS GISYSGGSKYYADSVKG AAVDFGRRAVRLDY + (2) (28) (49) GC5B444GC5H207 W101F NYWMS GISYSGGSKYYADSVKG AAFDFGRRAVRLDY + (2) (28) (97)GC5B464 GC5H206 W101G NYWMS GISYSGGSKYYADSVKG AAGDFGRRAVRLDY + (2) (28)(50) GC5B445 GC5H205 W101A NYWMS GISYSGGSKYYADSVKG AAADFGRRAVRLDY + (2)(28) (51) GC5B446 GC5H204 D62A NYWMS GISYSGGSKYYAASVKG AAWDFGRRAVRLDY +(2) (44) (30) GC5B447 GC5H203 D62S NYWMS GISYSGGSKYYASSVKGAAWDFGRRAVRLDY + (2) (6) (30) GC5B448 GC5H202 D62K NYWMSGISYSGGSKYYAKSVKG AAWDFGRRAVRLDY + (2) (45) (30) GC5B449 GC5H201 D62ENYWMS GISYSGGSKYYAESVKG AAWDFGRRAVRLDY + (2) (46) (30) GC5B450 GC5H200D62Y NYWMS GISYSGGSKYYAYSVKG AAWDFGRRAVRLDY + (2) (47) (30)

On the basis of this binding data, selected mAbs were generated asGPRC5D×CD3 bispecific antibodies and assessed for T-cell mediatedcytotoxicity of H929 cells (Table 18).

TABLE 18 Functional Activity of selected GPRC5D PTM variants for GCDB164and GCDB285 Parent GPRC5D GPRC5D Protein AA GPRC5D × H929 EC₅₀ Selectedas mAb ID ID CD3 ID (nM) Lead GC5B285 GC5B432 GCDB50 1.3 GC5B433 GCDB510.81 GC5B434 GCDB52 2.81 GC5B465 GCDB53 0.24 XX GC5B463 GCDB54 3.09GC5B164 GC5B471 GCDB57 3.87 GC5B476 GCDB58 1.24 GC5B478 GCDB59 1.54GC5B481 GCDB60 2.94 GC5B483 GCDB61 1.51 XX GC5B493 GCDB62 3.1

A range of potency was observed it the T-cell mediated cytotoxicityassay that was not necessarily predicted by the observed bindingaffinity. For example, GC5B465 and GC5B463 bound with similar affinityto human GPRC5D differ only in the sequence of 2 amino acids (Table 17)and were observed to have a 12.5 fold difference in potency asGPRC5D×CD3 bispecific Abs (Table 18). On the basis of the functionaldata GC5B465 and GC5B483 were selected as optimized sequences forGC5B285 (GCDB43 as CD3 bispecific) and GC5B164 (GCDB35 as CD3bispecific).

Human framework adaptation was also completed for the murine hybridomaderived GPCR5D mAbs (GC5B390 and GC5B396, or GCDB46 and 47 as CD3bispecific, respectively). Binding studies identified a number offrameworks for GC5B396 and one framework for GC5B390 which retainedbinding to human GPRC5D (Table 19).

TABLE 19 Binding and functional data for the human framework adaptationof hybridoma derived anti-GPRC5D mAb libraries. Binding was classifiedas MFI > 10,000 = +++; MFI > 5,000 = ++; MFI > 1,000 = +; MFI < 1,000.GPRC5D Human H929 Parent GPRC5D Heavy Light GPRC5D GPRC5D × EC50 mAb IDmAb AA ID Chain ID Chain ID (FACS) CD3 ID (nM) GPRC5D GC5B541 GC5H241GC5L58 + B396 GC5B540 GC5H240 GC5L58 +++ GCDB69 0.58 GC5B539 GC5H242GC5L58 ++ GC5B538 GC5H243 GC5L58 ++ GC5B537 GC5H241 GC5L57 + GC5B536GC5H240 GC5L57 +++ GCDB68 2.51 GC5B535 GC5H242 GC5L57 + GC5B534 GC5H243GC5L57 ++ GC5B533 GC5H241 GC5L56 + GC5B532 GC5H240 GC5L56 +++ GCDB670.61 GC5B531 GC5H242 GC5L56 ++ GC5B530 GC5H243 GC5L56 +++ GCDB66 1.41GC5B529 GC5H241 GC5L55 ++ GC5B528 GC5H240 GC5L55 +++ GCDB65 1.01 GC5B527GC5H242 GC5L55 − GC5B526 GC5H243 GC5L55 ++ GCDB64 1.5  GPRC5D GC5B525GC5H279 GC5L53 − B390 GC5B524 GC5H237 GC5L53 − GC5B523 GC5H238 GC5L53 −GC5B522 GC5H236 GC5L53 − GC5B521 GC5H279 GC5L52 − GC5B520 GC5H237 GC5L52− GC5B519 GC5H238 GC5L52 − GC5B518 GC5H236 GC5L52 − GC5B517 GC5H279GC5L51 + GC5B516 GC5H237 GC5L51 + GC5B515 GC5H238 GC5L51 ++ GCDB63 0.52GC5B514 GC5H236 GC5L51 + GC5B513 GC5H279 GC5L50 − GC5B512 GC5H237GC5L50 + GC5B511 GC5H238 GC5L50 + GC5B510 GC5H236 GC5L50 + GC5B509GC5H279 GC5L49 − GC5B508 GC5H237 GC5L49 + GC5B507 GC5H238 GC5L49 +GC5B506 GC5H236 GC5L49 +

On the basis of the binding data several anti-GPCR5D mAbs were generatedas CD3 bispecific antibodies and assessed for T-cell mediatedcytotoxicity of H929 cells (Table 18). Functional analysis identifiedGCDB63, GCDB67, and GCDB69 as potent fully humanized GPRC5D×CD3bispecific antibodies. On the basis of these data, the correspondinganti-GPRC5D mAbs, GC5B515, GC5B532, and GC5B540 were selected as thefully humanized sequences for GC5B390 and GC5B391.

Additional lead optimization of the fully humanized sequences was thencompleted aimed at addressing potential post-translation modificationsequence risks for GC5B515, GC5B532, and GCDB540. A G56S mutation wasgenerated in the heavy chain sequence to remove a potential deamidationrisk for GC5B515 (Table 20).

TABLE 20 Binding and Functional Activity of selected GPRC5D PTM variantsfor GC5B532, GC5B540 and GCDB515. GPRC5D GPRC5D Human H929 Parent mAb AAGPRC5D GPRC5D × EC50 mAb ID ID Mutation (FACS) CD3 ID (nM) GC5B540GC5B590 M64K +++ GC5B592 G99A + GC5B594 M64K, G99A + GC5 B532 GC5B591M64K +++ GC5B593 G99A + GC5B595 M64K, G99A + GC5 B515 GC5B596 G56S ++GCDB72 0.15

The heavy chains for GC5B532 and GC5B540 contained both a potentialisomerization and an oxidation risk. M64K and G99A mutations weregenerated to ameliorate this risk (Table 20). All of the variants testedwith the G99A mutation had a substantial reduction in binding affinity,while the M64K and G56A variants were not impacted. Based on the bindingdata alone GC5B596 was progressed to functional assessment anddemonstrated potency as a CD3 bispecific (GCDB72) in T-cell mediatedcytotoxicity assays.

Thus, four GPRC5D bispecific mAbs were selected for additionalcharacterization: GCDB32, GCDB53, GCDB61, and GCDB72. Depicted below inTables 21 and 22 are the CDR and heavy and light chain sequences of theGPRC5D mAbs used for the generation of the bispecific molecules.

TABLE 21CDR sequences of 4 GPRC5D mAb candidates that showed binding against human and cyno GPRC5D and that were functional when generated as CD3 bispecifics. ID HC-CDR1HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 GC5B81 SYAIS GIIPIFGTANYAQKFQGESRWRGYKLD  RASQSISSYLN AASSLQS QQSYSTPLT (SEQ ID NO 1) (SEQ ID NO 5)(SEQ ID NO 9) (SEQ ID NO 13) (SEQ ID NO 16) (SEQ ID NO 19) GC5B465 NYWMSGISYSGGSKYYASSVKG AAFDFGRRAVRLD RASQSISSYLN AASSLQS QQSYSTPLT(SEQ ID NO 2) (SEQ ID NO 6) (SEQ ID NO 10) (SEQ ID NO 13) (SEQ ID NO 16)(SEQ ID NO 19) GC5B483 SYFIG IIYPGKSDTRYSPSFQG VYSFGGRHKALFDYRASQSVSSYLA DASNRAT QQRSNWPLT (SEQ ID NO 3) (SEQ ID NO 7) (SEQ ID NO 11)(SEQ ID NO 14) (SEQ ID NO 17) (SEQ ID NO 20) GC5B596 GYTMNLINPYNSDTNYAQKLQG VALRVALDY  KASQNVATHVG SASYRYS QQYNRYPYT (SEQ ID NO 4)(SEQ ID NO 8) (SEQ ID NO 12) (SEQ ID NO 15) (SEQ ID NO 18)(SEQ ID NO 21)

TABLE 22 Heavy and light chain variable region sequences of 4 GPRC5D mAb candidates that showed binding against human and cyno GPRC5D and that werefunctional when generated as CD3 bispecifics. SEQ SEQ mAb AAVH Amino Acid ID VL Amino Acid ID ID Sequence NO: Sequence NO GC5B81QVQLVQSGAEVKK 52 DIQMTQSPSSLSAS 56 PGSSVKVSCKASGG VGDRVTITCRASQTFSSYAISWVRQAP SISSYLNWYQQKP GQGLEWMGGIIPIF GKAPKLLIYAASSL GTANYAQKFQGRVQSGVPSRFSGSGSG TITADESTSTAYME TDFTLTISSLQPEDF LSSLRSEDTAVYYCATYYCQQSYSTPLT ARESRWRGYKLDY FGQGTKVEIK WGQGTLVTVSS GC5B465EVQLLESGGGLVQP 53 DIQMTQSPSSLSAS 56 GGSLRLSCAASGFT VGDRVTITCRASQFSNYWMSWVRQAP SISSYLNWYQQKPG GKGLEWVSGISYSG KAPKLLIYAASSLQGSKYYASSVKGRFT SGVPSRFSGSGSGT ISRDNSKNTLYLQM DFTLTISSLQPEDFANSLRAEDTAVYYCA TYYCQQSYSTPLTF KAAFDFGRRAVRLD GQGTKVEIK YWGQGTLVTVSSGC5B483 EVQLVQSGAEVKKP 54 EIVLTQSPATLSLSP 57 GESLKISCKGSGYSFGERATLSCRASQS TSYFIGWVRQMPGK VSSYLAWYQQKPG GLEWMGIIYPGKSD QAPRLLIYDASNRATRYSPSFQGQVTISA TGIPARFSGSGSGT DKSISTAYLQWSSLK DFTLTISSLEPEDFAASDTAMYYCARVY VYYCQQRSNWPLT SFGGRHKALFDYWG FGQGTKVEIK QGTLVTVSS GC5B596QVQLVQSGAEVKK 55 DIQMTQSPSSLSAS 58 PGASVKVSCKASGY VGDRVTITCKASQNSFTGYTMNWVRQA VATHVGWYQQKPG PGQGLEWMGLINPY KAPKRLIYSASYRY NSDTNYAQKLQGRSGVPSRFSGSGSGTE VTMTTDTSTSTAYM FTLTISNLQPEDFAT ELRSLRSDDTAVYYYYCQQYNRYPYTF CARVALRVALDYW GQGTKLEIK GQGTLVTVSS

Example 7: Preparation of GPRC5D and CD3 Antibodies in a BispecificFormat in IgG4 S228P, L234A, L235A

The four monospecific GPRC5D antibodies (see table 21) were expressed asIgG4, having Fc substitutions S228P, L234A, and L235A or S228P, L234A,L235A, F405L, and R409K (CD3 arm) (numbering according to EU index). Amonospecific anti-CD3 antibody CD3B219 was also generated comprising theVH and VL regions having the heavy chain of SEQ ID NO: 25 and the lightchain of SEQ ID NO: 26 and IgG4 constant region with S228P, L234A,L235A, F405L, and R409K substitutions.

The monospecific antibodies were purified using standard methods using aProtein A column (HiTrap MabSelect SuRe column). After elution, thepools were dialyzed into D-PBS, pH 7.2.

Bispecific GPRC5D×CD3 antibodies were generated by combining amonospecific CD3 mAb and a monospecific GPRC5D mAb in in-vitro Fab armexchange (as described in WO2011/131746). Briefly, at about 1-20 mg/mLat a molar ratio of 1.08:1 of anti-GPRC5D/anti-CD3 antibody in PBS, pH7-7.4 and 75 mM 2-mercaptoethanolamine (2-MEA) was mixed together andincubated at 25-37° C. for 2-6 hours, followed by removal of the 2-MEAvia dialysis, diafiltration, tangential flow filtration and/or spin cellfiltration using standard methods.

Heavy and Light chains for the GPRC5D×CD3 bispecific Abs are shown belowin Table 23.

TABLE 23 Heavy and Light Chain Sequences for bispecific  Abs IgG4-PAA AbAmino Acid Sequence GCDB32 Heavy  EVQLVESGGGLVQPGGSLRLSCAASGFTFN chain 1TYAMNWVRQAPGKGLEWVARIRSKYNNY CD3B219 ATYYAASVKGRFTISRDDSKNSLYLQMNSL(SEQ ID KTEDTAVYYCARHGNFGNSYVSWFAYWG NO: 25) QGTLVTVSS Light QTVVTQEPSLTVSPGGTVTLTCRSSTGAVT Chain 1 TSNYANWVQQKPGQAPRGLIGGTNKRAPGCD3B219 TPARFSGSLLGGKAALTLSGVQPEDEAEYY (SEQ ID CALWYSNLWVFGGGTKLTVLNO: 26) Heavy  QVQLVQSGAEVKKPGSSVKVSCKASGGTFS chain 2SYAISWVRQAPGQGLEWMGGIIPIFGTANY GC5B81 AQKFQGRVTITADESTSTAYMELSSLRSEDT(SEQ ID AVYYCARESRWRGYKLDYWGQGTLVTVSS NO: 52) Light DIQMTQSPSSLSASVGDRVTITCRASQSISSY Chain 2 LNWYQQKPGKAPKLLIYAASSLQSGVPSRFGC5B81 SGSGSGTDFTLTISSLQPEDFATYYCQQSYS (SEQ ID TPLTFGQGTKVEIK NO: 56)GCDB53 Heavy  EVQLVESGGGLVQPGGSLRLSCAASGFTFNT chain 1YAMNWVRQAPGKGLEWVARIRSKYNNYAT CD3B219 YYAASVKGRFTISRDDSKNSLYLQMNSLKTE(SEQ ID DTAVYYCARHGNFGNSYVSWFAYWGQGTL NO: 25) VTVSS Light QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTS Chain 1 NYANWVQQKPGQAPRGLIGGTNKRAPGTPACD3B219 RFSGSLLGGKAALTLSGVQPEDEAEYYCALW (SEQ ID YSNLWVFGGGTKLTVL NO: 26)Heavy  EVQLLESGGGLVQPGGSLRLSCAASGFTFSNY chain 2WMSWVRQAPGKGLEWVSGISYSGGSKYYAS GC5B465 SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV(SEQ ID YYCAKAAFDFGRRAVRLDYWGQGTLVTVSS NO: 53) Light DIQMTQSPSSLSASVGDRVTITCRASQSISSYL Chain 2NWYQQKPGKAPKLLIYAASSLQSGVPSRFSG GC5B465 SGSGTDFTLTISSLQPEDFATYYCQQSYSTPL(SEQ ID TFGQGTKVEIK NO: 56) GCDB61 Heavy EVQLVESGGGLVQPGGSLRLSCAASGFTFNT chain 1 YAMNWVRQAPGKGLEWVARIRSKYNNYATCD3B219 YYAASVKGRFTISRDDSKNSLYLQMNSLKTE (SEQ IDDTAVYYCARHGNFGNSYVSWFAYWGQGTL NO: 25) VTVSS Light QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTS Chain 1 NYANWVQQKPGQAPRGLIGGTNKRAPGTPACD3B219 RFSGSLLGGKAALTLSGVQPEDEAEYYCAL (SEQ ID WYSNLWVFGGGTKLTVL NO: 26)Heavy  EVQLVQSGAEVKKPGESLKISCKGSGYSFTS chain 2YFIGWVRQMPGKGLEWMGIIYPGKSDTRYS GC5B483 PSFQGQVTISADKSISTAYLQWSSLKASDTA(SEQ ID MYYCARVYSFGGRHKALFDYWGQGTLVTVSS NO: 54) Light EIVLTQSPATLSLSPGERATLSCRASQSVSSY Chain 2 LAWYQQKPGQAPRLLTYDASNRATGIPARFGC5B483 SGSGSGTDFTLTISSLEPEDFAVYYCQQRSN (SEQ ID WPLTFGQGTKVEIK NO: 57)GCDB72 Heavy  EVQLVESGGGLVQPGGSLRLSCAASGFTFNT chain 1YAMNWVRQAPGKGLEWVARIRSKYNNYAT CD3B219 YYAASVKGRFTISRDDSKNSLYLQMNSLKTE(SEQ ID DTAVYYCARHGNFGNSYVSWFAYWGQGTL NO: 25) VTVSS Light QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTS Chain 1 NYANWVQQKPGQAPRGLIGGTNKRAPGTPACD3B219 RFSGSLLGGKAALTLSGVQPEDEAEYYCAL (SEQ ID WYSNLWVFGGGTKLTVL NO: 26)Heavy  QVQLVQSGAEVKKPGASVKVSCKASGYSFT chain 2GYTMNWVRQAPGQGLEWMGLINPYNSDTN GC5B596 YAQKLQGRVTMTTDTSTSTAYMELRSLRSD(SEQ ID DTAVYYCARVALRVALDYWGQGTLVTVSS NO: 55) Light DIQMTQSPSSLSASVGDRVTITCKASQNVAT Chain 2 HVGWYQQKPGKAPKRLIYSASYRYSGVPSRGC5B596 FSGSGSGTEFTLTISNLQPEDFATYYCQQYN (SEQ ID RYPYTFGQGTKLEIK NO: 58)

Example 8: Functional Characterization of GCDB32, GCDB53, GCDB61, andGCDB72

GCDB32, GCDB53, GCDB61, and GCDB72 were assessed for binding to murineGPRC5D (Table 24). All four bispecific antibodies bound to murineGPRC5D, with a range of binding affinities observed.

TABLE 24 Binding of anti-GPCR5D × CD3 antibodies to murine GPRC5D MurineGPRC5D × GPRC5D FACS CD3 ID binding (MFI) GCDB32 1151 GCDB53 2658 GCDB613552 GCDB72 481

Cross-reactivity with cyno GPRC5D was also assessed using the T-cellredirection cytotoxicity assay of over-expressed human and cyno GPRC5Dcell lines (Table 25). One GPRC5D×CD3 bispecific antibody was equipotentagainst human and cyno GPRC5D (GCDB32), while the other bispecificstested were less potent at inducing cytotoxicity of cyno GPRC5D thanhuman GPRC5D.

TABLE 25 Functional activity of lead GPRC5D × CD3 antibodies againsthuman and cyno GPRC5D-expressing HEK cells. GPRC5D × Human GPRC5D CynoGPRC5D HEK CD3 ID HEK cells (EC₅₀ nM) cells (EC₅₀ nM) GCDB32 0.04 0.07GCDB53 0.08 0.36 GCDB61 0.03 1.22 GCDB72 0.03 3.41Additional characterization was aimed at understanding in vitro binding(FIGS. 11A-11E) and potency (Table 26).

TABLE 26 T-cell mediated cytotoxicity of several human GPRC5D-expressing B cell lines by lead GPRC5D × CD3 antibodies. H929 cells MM1ROPM2 LP-1 EC₅₀ nM EC₅₀ nM EC₅₀ nM EC₅₀ nM GPRC5D × (Rank (Rank (Rank(Rank CD3 ID order) order) order) Order) GCDB32 0.45 (3) 0.04 (1) 0.98(2) 1.02 (2) GCDB53 0.69 (4) 0.28 (4) 2.83 (4) 1.58 (3) GCDB61 0.39 (2)0.06 (3) 1.46 (3) 1.67 (4) GCDB72 0.22 (1) 0.04 (1) 0.77 (1)  0.7 (1)While a range of binding affinities were observed, with GCDB61 thestrongest binder and GCDB72 & GCDB32 the weakest binders, the in vitropotencies were very similar for the panel of bispecific antibodies.However, based on rank order analysis GCDB72 was the most potent acrossthe various B cell lines analyzed. Ex vivo binding and potencyexperiments using patient derived MNCs yielded larger similar results tothe in vitro assays (Table 27 and FIGS. 15A and 15B).

TABLE 27 GPCR5D × CD3 bispecific antibody binding, T-cell mediatedcytotoxicity, and T-cell activation of MM patient derived MNCs. GPRC5D ×Binding T cell CD3 EC₅₀ Cytotoxicity activation Patient AA ID nM EC₅₀ nMEC₅₀ nM MM303BM GCDB32 16.1 0.36 0.18 GCDB53 3.98 0.42 0.17 GCDB61 5.530.89 0.22 GCDB72 27.5 0.30 0.12 MM305BM GCDB32 70.4 0.26 0.16 GCDB5310.2 0.26 0.19 GCDB61 6.93 0.6 0.22 GCDB72 65.9 0.28 0.12GCDB61 had the highest binding affinity to patient MNCs while GCDB72 &GCDB32 were the weakest binders. Again, even though differences inbinding affinity were observed, all bispecific antibodies demonstratedsub-nanomolar efficacy in T-cell redirected cytotoxicity assays. Themolecules were virtually indistinguishable on the basis of in vitro andex vivo potency however, in vivo data provided differentiation (FIG.12A-12D).

H929 cells were implanted into NSG mice, one week following injection ofhuman PBMCs. Treatment of the bispecific antibodies was initiated at thesame time that the H929 cells were implanted and continued every 2 or 3days (q2d or q3d) at 10 ug, 1 ug, and 0.1 ug/animal doses for a total offive treatments. Ten mice were used in each group and PBS included asthe vehicle control. Treatment was stopped at day 11 and the study wasterminated on day 26 (FIG. 12 A-D). All the GPRC5D×CD3 bispecificmolecules tested in this prophylactic model showed 100% tumor growthinhibition at the 10 and 1 ug/animal dose. Differentiation was observedat the lowest dose, 0.1 ug/animal, with GCDB72 demonstrating superiorityto the other bispecific antibodies tested with 80% tumor growthinhibition observed.

Example 9: GPRC5D Antibody Binding Profile on GPRC5D⁺MM1.R Cell Line

The binding affinities of the GPRC5D antibodies on GPRC5D⁺ human MM cellline (MM1.R, purchased from ATCC (American Type Culture Collection))were measured using FACS. FIG. 13 shows that all the lead antibodiesbound to GPRC5D expressing MM.1R cells in a dose dependent manner withEC₅₀ values ranging from 0.10 nM to 135 nm all of which except GC5B602are significantly lower than the values of commercial antibody FAB6300(R& D Systems Cat No. FAB6300A, Clone No. 571961) which yielded an EC₅₀value of 121.7 nM.

GPRC5D⁺MM1.R cell lines were stained for 60 minutes with variousconcentrations of lead antibodies to measure the surface bindingprofiles (n=3). Phycoerythrin labelled human IgG4Fc was used as asecondary antibody to capture the signal (Southern Biotech, cloneHP6025, Cat #9200-09). Binding is expressed as normalized geometricalmean fluorescence intensity as determined by FACS. Graphing and fittingof data were done in GraphPad Prism 6 using nonlinear regression withvariable slope (four parameters) and least square fit method.

In addition, the GPRC5D mAb GC5M481 binding profile was assessed incomparison with the commercial antibody using three GPRC5D⁺ (JIM3,OPM-2, and MM.1R; Cell lines purchased from ATCC) multiple myeloma celllines (FIG. 14A). Further, the GPRC5D mAb (GC5M481) was profiled forcyno cross-reactivity using a cyno-GPRC5D expressing Daudi cells whichshowed strong binding compared to the parental cells (FIG. 14A). Also,five GPRC5D×CD3 bispecific antibodies (GCDB32, GCDB48, GCDB53, GCDB61and GCDB72) when evaluated for the binding potential using GPRC5D+(JIM3,OPM-2 and MM1.R) cell lines (FIG. 14B) showed a significant binding asevident by the shift in the histogram (black solid trace) compared tothe isotype control (dotted, gray filled trace).

Example 10: Anti-Tumor Efficacy of GCDB72 Against Subcutaneous MM.1SHuman Multiple Myeloma Xenografts in PBMC-Humanized NSG Mice

This in vivo study was performed to determine the efficacy of GCDB72against established MM.1S human multiple myeloma (MM) xenografts in PBMChumanized NSG mice. Female NSG mice of similar weight and age weresubcutaneously (sc) implanted with MM.1S human MM cells (1×10⁷ cells in200 μL PBS per mouse) on the right dorsal hind flank on study day 0. Onday 7 post tumor cell implantation, 1×10⁷ human PBMC (in 200 μL PBS)were injected intravenously via the lateral tail vein. Treatments wereinitiated on day 15 when mean tumor volume was approximately 72-78 mm³,each mouse received intravenous (iv) administration of PBS or GCDB72DuoBody antibody at 0.1 μg (0.005 mg/kg), 1 (0.05 mg/kg), 10 μg (0.5mg/kg) and 50 μg (2.5 mg/kg). Null DuoBody controls, CD3×Null andNull×GPRC5D, were each dosed at 10 μg per mouse. Treatments wereadministered approximately every three days (q3d) for a total of sevendoses. Robust anti-tumor efficacy was observed with the two high doses(10 μg and 50 μg) of GCDB72 where MM.1S sc tumors completely regressedin 100% (10 of 10 per group) of the animals by the end of the study(FIG. 16 ). Moreover, the 1 μg per mouse dose significantly inhibitedtumor growth by 65% (p≤0.0001) compared to PBS treated tumors, while the0.1 μg dose had little effect (TGI=19.3%, p=0.0023). The effect ofCD3×Null was not considered efficacious (TGI=28%, p≤0.0001) andNull×GPRC5D had negligible effect of 3.1% TGI, p=0.9971. TGI wasdetermined on day 36 when there at least 80% viable animals per group.Significant body weight loss and/or mortality began to manifest post day36 due to GVHD (FIG. 17 ). The study was terminated on day 43 when therewere 60% or less animals remaining in the groups.

Example 11: Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) ofGPRC5D Antibodies

A panel of anti-human GPRC5D mAbs were generated as IgG1 mAbs. Inaddition, a new panel of anti-human GPRC5D mAbs were generated asdescribed in Example 2. Depicted below in Tables 28 and 29 are the CDRand heavy and light chain variable region sequences of the new GPRC5DmAbs. These new antibodies were used for the generation of bispecificCD3 molecules as described in Example 7 and also incorporated as IgG1mAbs for ADCC activity assessment.

TABLE 28 CDR sequences of new panel of GPRC5D antibodies ID HC-CDR1HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 GC5B382 DYGMH AIKYSGGSTYYADSVKGRAESGPGLDY KSSQSVLYSSNNK WASTRES QQYYSTPLT (SEQ ID NO 61) (SEQ ID NO 67)(SEQ ID NO 72) NYLA (SEQ ID  (SEQ ID NO 78) (SEQ ID NO 80) NO 98)GC5B379 NYWMS GISYSGGSKYYADSVKG AAWDFGRRAVRLDY RASQSISSYLN AASSLQSQQSYSTPLT (SEQ ID NO 2) (SEQ ID NO 28) (SEQ ID NO 30) (SEQ ID NO 13)(SEQ ID NO 16) (SEQ ID NO 19) GC5B373 SYWIG IIYPGDSDTRYSPSFQGIGFYGRSFRIFDY RASQSVSSYLA DASNRAT QQRSNWPLT (SEQ ID NO 27)(SEQ ID NO 29) (SEQ ID NO 73) (SEQ ID NO 14) (SEQ ID NO 17)(SEQ ID NO 20) GC5B376 SYWIG IIYPGDSDTRYSPSFQG VYSFGGRHKALFDYRASQSVSSYLA DASNRAT QQRSNWPLT (SEQ ID NO 27) (SEQ ID NO 29)(SEQ ID NO 11) (SEQ ID NO 14) (SEQ ID NO 17) (SEQ ID NO 20) GC5B385GYAMS AISGSGGSTYYADSVKG VDRSFGRSRYTLDY RASQSVSSYLA DASNRAT QQRSNWPLT(SEQ ID NO 62) (SEQ ID NO 68) (SEQ ID NO 74) (SEQ ID NO 14)(SEQ ID NO 17) (SEQ ID NO 20) GC5B370 SYGIS GIIPIFGNINYAQKFQGVSRRFKRFAYYFDY KSSQSVLYSSNNK WASTRES QQYYSTPLT GC5B597 (SEQ ID NO 63)(SEQ ID NO 69) (SEQ ID NO 75) NYLA (SEQ ID  (SEQ ID NO 78)(SEQ ID NO 80) NO 98) GC5B602 GYSFTGYTMN LINPYNGDTN VALRVALDYKASQNVATHVG SASYRYS QQYNRYPYT (SEQ ID NO 64) (SEQ ID NO 70)(SEQ ID NO 12) (SEQ ID NO 15) (SEQ ID NO 18) (SEQ ID NO 21) GC5B603SYAMS AISGSGGSTYYADSVKG SNFLPVVFDY RASQSVRKSLA TASNRAT QQYFRAPIT(SEQ ID NO 65) (SEQ ID NO 68) (SEQ ID NO 76) (SEQ ID NO 95)(SEQ ID NO 79) (SEQ ID NO 81) GC5B601 GFSLTSYNVH VIWAGGSTNYNSALMSDGIRLRFAY KASQNVATHVG SASYRYS QQYNRYPYT (SEQ ID NO 66) (SEQ ID NO 71)(SEQ ID NO 77) (SEQ ID NO 15) (SEQ ID NO 18) (SEQ ID NO 21)

TABLE 29 Heavy and Light chain variable region sequencesof new panel of GPRC5D antibodies SEQ SEQ mAb AA VH Amino Acid IDVL Amino Acid ID ID Sequence NO: Sequence NO GC5B382 EVQLLESGGGLVQP 82DIVMTQSPDSLAVS 92 GGSLRLSCAASGFT LGERATINCKSSQS FSDYGMHWVRQAPVLYSSNNKNYLAW GKGLEWVSAIKYSG YQQKPGQPPKLLIY GSTYYADSVKGRFTWASTRESGVPDRFS ISRDNSKNTLYLQM GSGSGTDFTLTISSL NSLRAEDTAVYYCQAEDVAVYYCQQY AKRAESGPGLDYW YSTPLTFGQGTKVE GQGTLVTVSS IK GC5B379EVQLLESGGGLVQP 83 DIQMTQSPSSLSAS 56 GGSLRLSCAASGFT VGDRVTITCRASQSFSNYWMSWVRQAP ISSYLNWYQQKPGK GKGLEWVSGISYSG APKLLIYAASSLQSGGSKYYADSVKGRF VPSRFSGSGSGTDFT TISRDNSKNTLYLQ LTISSLQPEDFATYYMNSLRAEDTAVYY CQQSYSTPLTFGQG CAKAAWDFGRRA TKVEIK VRLDYWGQGTLVT VSSGC5B373 EVQLVQSGAEVKKP 84 EIVLTQSPATLSLSP 57 GESLKISCKGSGYSFGERATLSCRASQSV TSYWIGWVRQMPG SSYLAWYQQKPGQ KGLEWMGIIYPGDS APRLLIYDASNRATDTRYSPSFQGQVTIS GIPARFSGSGSGTD ADKSISTAYLQWSS FTLTISSLEPEDFAVLKASDTAMYYCAR YYCQQRSNWPLTF IGFYGRSFRIFDYW GQGTKVEIK GQGTLVTVSS GC5B376EVQLVQSGAEVKKP 85 EIVLTQSPATLSLSP 57 GESLKISCKGSGYSF GERATLSCRASQSVTSYWIGWVRQMPG SSYLAWYQQKPGQ KGLEWMGIIYPGDS APRLLIYDASNRATDTRYSPSFQGQVTIS GIPARFSGSGSGTD ADKSISTAYLQWSS FTLTISSLEPEDFAVLKASDTAMYYCAR YYCQQRSNWPLTF VYSFGGRHKALFD GQGTKVEIK YWGQGTLVTVSS GC5B385EVQLLESGGGLVQP 86 EIVLTQSPATLSLSP 57 GGSLRLSCAASGFT GERATLSCRASQSVFSGYAMSWVRQAP SSYLAWYQQKPGQ GKGLEWVSAISGSG APRLLIYDASNRAT GSTYYADSVKGRFTGIPARFSGSGSGTDF ISRDNSKNTLYLQM TLTISSLEPEDFAVY NSLRAEDTAVYYCYCQQRSNWPLTFG AKVDRSFGRSRYTL QGTKVEIK DYWGQGTLVTVSS GC5B370QVQLVQSGAEVKKP 87 DIVMTQSPDSLAVS 92 GC5B597 GSSVKVSCKASGGTLGERATINCKSSQS FSSYGISWVRQAPG VLYSSNNKNYLAW QGLEWMGGIIPIFGNYQQKPGQPPKLLIY INYAQKFQGRVTITA WASTRESGVPDRFS DESTSTAYMELSSLRGSGSGTDFTLTISSL SEDTAVYYCARVSR QAEDVAVYYCQQY RFKRFAYYFDYWGYSTPLTFGQGTKVE QGTLVTVSS IK GC5B602 QVQLVQSGAEVKK 88 DIQMTQSPSSLSAS 58PGASVKVSCKASG VGDRVTITCKASQN YSFTGYTMNWVRQ VATHVGWYQQKPG APGQGLEWMGLINKAPKRLIYSASYRY PYNGDTNYAQKLQ SGVPSRFSGSGSGTE GRVTMTTDTSTSTAFTLTISNLQPEDFAT YMELRSLRSDDTA YYCQQYNRYPYTFG VYYCARVALRVAL QGTKLEIKDYWGQGTLVTVSS GC5B603 EVQLLESGGGLVQP 89 EIVLTQSPATLSLSP 93GGSLRLSCAASGFT GERATLSCRASQSV FSSYAMSWVRQAP RKSLAWYQQKPGQ GKGLEWVSAISGSGAPRLLIYTASNRATG GSTYYADSVKGRF IPARFSGSGSGTDFTL TISRDNSKNTLYLQTISSLEPEDFAVYYC MNSLRAEDTAVY QQYFRAPITFGQGTK YCAKSNFLPVVFD VEIKKYWGQGTLVTVSS GC5B601 QVTLKESGPVLVKP 91 EIVMTQSPATLSVSP 94 TETLTLTCTVSGFSGERATLSCKASQNV LTSYNVHWIRQPPG ATHVGWYQQKPGQ KALEWLAVIWAGGAPRLLIYSASYRYSG STNYNSALMSRLTI IPARFSGSGSGTEFTL SKDTSKSQVVLTMTISSLQSEDFAVYYC TNMRAEDTATYYC QQYNRYPYTFGQGTK ARDGIRLRFAYWG LEIKQGTLVTVSSADCC activity against H929 cells (Tables 30 and 31). Briefly, multiplemyeloma cells were labeled with Calcein-AM for 30 minutes at roomtemperature and re-suspend at 0.2×10⁶/ml in RPMI+10% HI FBS after twoPBS washes. PBMC's were thawed and resuspended after PBS wash at 3×10⁶cells per ml in RPMI growth media. 10000 or 50000 target cells weremixed with 100000 or 2500000 PBMC'S in presence of the antibody andincubated for 3 hours in CO2 incubator at 37° C. After the incubationplate was centrifuges at 200 g for 4 minutes and 100 ul of supernatantwas transferred into a new 96 well plate and the fluorescence intensitywas measured at 485/535 nM. RFU values were plotted to calculate thepercent lysis

TABLE 30 Antibody dependent cytotoxicity of H929 cells by anti-GPRC5DmAbs with IgG1 Fc. GPRC5D IgG4 GPRC5D H929 Protein IgG1 Cytotoxicity %ID Protein ID EC₅₀ (pM) Lysis GC5B243 GC5B382 1346.5 29.14 GC5B285GC5B379 87.4 17.3 GC5B332 GC5B373 244.2 32.94 GC5B164 GC5B376 22.0 32.21GC5B320 GC5B385 944.0 29.73 GC5B251 GC5B370 24.2 8 GC5B515 GC5B60227721.7 20 GC5B420 GC5B603 3.4 16 GC5B483 GC5B599 7.4 14 GC5B540 GC5B601169.6 14 GC5B465 GC5B598 2.0 12 GC5B251 GC5B597 705.1 10

TABLE 31 Comparison of Antibody dependent cytotoxicity and T-cellmediated cytotoxicity of H929 cells. GPRC5D × ADCC CD3 RedirectionGPRC5D CD3 H929 T-cell Mediated H929 IgG1 DuoBody CytotoxicityCytotoxicity EC50 Protein ID ID EC50 (pM) (nM) GC5B382 GCDB40 1346.5 1.2 ± 0.83 GC5B379 GCDB43 87.4 0.39 ± 0.07 GC5B373 GCDB45 244.2 2.36 ±0.84 GC5B376 GCDB35 22.0 1.39 ± 0.75 GC5B385 GCDB44 944.0 >10 GC5B370GCDB41 24.2 >10 GC5B602 GCDB72 27721.7 0.15 GC5B603 GCDB48 3.4 0.17GC5B599 GCDB61 7.4 1.51 GC5B601 GCDB69 169.6 0.58 GC5B598 GCDB53 2.00.24A range of potency was observed ranging from 2 pM to 27.7 nM. Bindingaffinity was not necessarily predictive of efficacy in the ADCC assay.For example, GC5B382 and GC5B379 had similar binding affinity to humanGPRC5D cells but a 15× difference in cytotoxicity against H929 cells inthe ADCC assay. Similarly, cytotoxic inducement as a GPRC5D×CD3bispecific was not predictive of potency in the ADCC assay asexemplified by GC5B370 and GC5B602. When formatted as a CD3 bispecificGC5B602 (GCDB63) had sub-nanomolar potency against H929 cells whileGC5B370 as a CD3 bispecific (GCDB41) was essentially inactive. The samev-regions, when formatted as IgG1 mAbs resulted in the oppositeobservation in the ADCC assay, with GC5B370 observed as more potent (by−1100× fold) over GC5B602.

Brief Description of the Sequence Listing SEQ ID NO: Type SpeciesDescription Sequence  1 PRT human GC5B81, GC5B427, SYAISGC5B428, GC5B430, GC5B431, AND GC5B429-HCDR1  2 PRT humanGC5B379, GC5B598, NYWMS GC5B465, GC5B285, GC5B463, GC5B432,GC5B433, GC5B434, GC5B435, GC5B436, GC5B461, GC5B462, GC5B437, GC5B438,GC5B439, GC5B440, GC5B441, GC5B442, GC5B443, GC5B444, GC5B464, GC5B445,GC5B446, GC5B447, GC5B448, GC5B449, and GC5B450-HCDR1  3 PRT humanGC5B483, GC5B473,  SYFIG GC5B478, GC5B488,  GC5B493, and GC5B498-HCDR1 4 PRT human GC5B596-HCDR1 GYTMN  5 PRT human GC5B81, GC5B427,GIIPIFGTANYAQKFQG GC5B428, GC5B430, GC5B431, and GC5B429-HCDR2  6 PRThuman GC5B598, GC5B465, GISYSGGSKYYASSVKG GC5B432, AND GC5B447-HCDR2  7PRT human GC5B599, GC5B483, IIYPGKSDTRYSPSFQG GC5B481, GC5B482,GC5B484, GC5B485, AND GC5B503-HCDR2  8 PRT human GC5B596-HCDR2LINPYNSDTNYAQKLQG  9 PRT human GCB581-HCDR3 ESRWRGYKLD 10 PRT humanGC5B598, GCB5465- AAFDFGRRAVRLD HCDR3 11 PRT human GC5B376, GC5B599,VYSFGGRHKALFDY GC5B483, GC5B164, GC5B471, GC5B472, GC5B473, GC5B474,GC5B475, GC5B476, GC5B477, GC5B478, GC5B479, GC5B480, GC5B481, GC5B482,GC5B484, GC5B485, GC5B486, GC5B487, GC5B488, GC5B489, GC5B490, GC5B491,GC5B492, GC5B493, GC5B494, GC5B495, GC5B496, GC5B497, GC5B498, GC5B499,GC5B500, GC5B501, GC5B502, GC5B503, GC5B504, AND GC5B505-HCDR3 12 PRThuman GC5B602, GCB596- VALRVALDY HCDR3 13 PRT human GC5B382, GC5B379,RASQSISSYLN GC5B370, GC5B598, GC5B597, GC5B81, GC5B465-LCDR1 14 PRThuman GC5B373, GC5B376, RASQSVSSYLA GC5B385, GC5B599, GC5B483-LCDR1 15PRT human GC5B605, GC5B601, KASQNVATHVG GC5B596-LCDR1 16 PRT humanGC5B81, GC5B465, AASSLQS GC5B379, GC5B598- LCDR2 17 PRT humanGC5B373, GC5B376, DASNRAT GC5B385, GC5B599, GC5B483-LCDR2 18 PRT humanGC5B602, GC5B601, SASYRYS GC5B596-LCDR2 19 PRT human GC5B379, GC5B598,QQSYSTPLT GC5B81, GC5B465- LCDR3 20 PRT human GC5B373, GC5B376,QQRSNWPLT GC5B385, GC5B599, GC5B483-LCDR3 21 PRT human GC5B602, GC5B601,QQYNRYPYT GC5B596-LCDR3 22 PRT human GPRC5D MYKDCIESTGDYFLLCDAEGPWGIILESLAILGIVVTILLLLAFLFLMRKIQDCSQW NVLPTQLLFLLSVLGLFGLAFAFIIELNQQTAPVRYFLFGVL FALCFSCLLAHASNLVKLVRGCVSFSW TTILCIAIGCSLLQIIIATEYVTLIMTRGMMFVNMTPCQLNV DFVVLLVYVLFLMALTFFVSKATFCGP CENWKQHGRLIFITVLFSIIIWVVWISMLLRGNPQFQRQPQWD DPVVCIALVTNAWVFLLLYIVPELCILY RSCRQECPLQGNACPVTAYQHSFQVENQELSRARDSDGAEE DVALTSYGTPIQPQTVDPTQECFIPQAK LSPQQDAGGV 23PRT cyno GPRC5D MYKDCIESTGDYFLPCDSEGPWGIVLESLAILGIVVTILLLLAFLFLMRKIQDCSQW NVLPTQLLFLLSV LGLFGLAFAFIIQLNQQTAPVRYFLFGVLFALCFSCLLAHASNLVKLVRGRVSFS WTTILCIAIGCSLLQVIIAIEYVTLIMTRGMMFVHMTPYQLNVDFVVLLVYVLFLM ALTFFVSKATFCGPCENWKQHGRLIFITVLFSIIIWVVWISMLLRGNPQFQRQPQW DDPVVCIALVTNA WVFLLLYIVPELCILYRSCRQECPSQGHACPVTAYQRSFQVENQELSRARDSDGA EEDVALTSFGTPIQPQTVDPTQECFIPRA KLSPQQDAGV 24PRT mouse GPRC5D MYEDCVKSTEDYYLFCDNEGPWAIVLESLAVIGIVVTILLLLAFLFLMRKVQDCS QWNVLPTQFLFLLAVLGLFGLTFAFIIQLNHQTAPVRYFLFGVL FAICFSCLLAHASNLVKLVRGRVSFCWT TILFIAIGVSLLQTIIAIEYVTLIMTRGLMFEHMTPYQLNV DFVCLLIYVLFLMALTFFVSKATFCGPC ENWKQHGRLIFATVLVSIIIWVVWISMLLRGNPQLQRQPHW DDAVICIGLVTNAWVFLLIYIIPELSILYR SCRQECPTQGNVCQVPVYQRSFRMDTQEPTRARDSDGAQE DVALTAYGTPIQLQSADPSREYLIPSATL SPQQDAGL 25PRT human CD3B219- EVQLVESGGGLVQPGGSLRLSCAASGF HEAVY CHAINTFNTYAMNWVRQAPGKGLEWVARIRS KYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGN SYVSWFAYWGQGTLVTVSS 26 PRT human CD3B219-QTVVTQEPSLTVSPGGTVTLTCRSSTGA LIGHT CHAIN VTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCALWYSNLWVFGGGTKLTVL 27 PRT humanGC5B373, GC5B376, SYWIG GC5B164, GC5B501, GC5B502, GC5B503, GC5B504, ANDGC5B505-HCDR1 28 PRT human GC5B379, GC5B285, GISYSGGSKYYADSVKGGC5B442, GC5B443, GC5B444, GC5B464, AND GC5B445-HCDR2 29 PRT humanGC5B373, GC5B376, IIYPGDSDTRYSPSFQG GC5B164, GC5B496, GC5B497, GC5B498,GC5B499, AND GC5B500-HCDR2 30 PRT human GC5B379, GC5B285, AAWDFGRRAVRLDYGC5B446, GC5B447, GC5B448, GC5B449, AND GC5B450-HCDR3 31 PRT humanGC5B427-HCDR3 ESRYRGYKLDY 32 PRT human GC5B428-HCDR3 ESRVRGYKLDY 33 PRThuman GC5B430-HCDR3 ESRGRGYKLDY 34 PRT human GC5B431-HCDR3 ESRARGYKLDY35 PRT human GC5B429 HCDR3-VH ESRFRGYKLDY 36 PRT human GC5B471, GC5B476,SYYIG GC5B486, GC5B481, GC5B491, and GC5B496,-HCDR1 37 PRT humanGC5B497, GC5B472, SYVIG GC5B477, GC5B482, GC5B487, AND GC5B492-HCDR1 38PRT human GC5B474, GC5B479, SYGIG GC5B484, GC5B489, GC5B494, ANDGC5B499-HCDR1  39 PRT human GC5B475, GC5B480,  SYAIG GC5B485, GC5B490, GC5B495, AND GC5B500-HCDR1 40 PRT human GC5B471, GC5B472,IIYPGASDTRYSPSFQG GC5B473, GC5B474, GC5B475, AND GC5B501-HCDR2 41 PRThuman GC5B476, GC5B477, IIYPGSSDTRYSPSFQG GC5B478, GC5B479, GC5B480, ANDGC5B502-HCDR2 42 PRT human GC5B486, GC5B487, IIYPGESDTRYSPSFQGGC5B488, GC5B489, GC5B490, AND GC5B504-HCDR2 43 PRT humanGC5B491, GC5B492, IIYPGYSDTRYSPSFQG GC5B493, GC5B494, GC5B495, ANDGC5B505-HCDR2 44 PRT human GC5B463 AND GISYSGGSKYYAASVKG GC5B446-HCDR245 PRT human GC5B433, GC5B434, GISYSGGSKYYAKSVKG AND GC5B448-HCDR2 46PRT human GC5B435, GC5B436, GISYSGGSKYYAESVKG GC5B461, GC5B462,AND GC5B449-HCDR2 47 PRT human GC5B437, GC5B438, GISYSGGSKYYAYSVKGGC5B439, GC5B440, GC5B441, AND GC5B450-HCDR2 48 PRT humanGC5B463, GC5B435, AAYDFGRRAVRLDY GC5B437, AND GC5B442-HCDR3 49 PRT humanGC5B432, GC5B433, AAVDFGRRAVRLDY GC5B438, AND GC5B443-HCDR3 50 PRT humanGC5B461, GC5B440, AAGDFGRRAVRLDY AND GC5B464-HCDR3 51 PRT humanGC5B462,GC5B441, AAADFGRRAVRLDY AND GC5B445-HCDR3 52 PRT human GC5B81-VHQVQLVQSGAEVKKPGSSVKVSCKASGG TFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYME LSSLRSEDTAVYYCARESRWRGYKLDY WGQGTLVTVSS 53PRT human GC5B598,  EVQLLESGGGLVQPGGSLRLSCAASGFT GC5B465-VHFSNYWMSWVRQAPGKGLEWVSGISYS GGSKYYASSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAAFDFGRRA VRLDYWGQGTLVTVSS 54 PRT human GC5B599,EVQLVQSGAEVKKPGESLKISCKGSGYS GC5B483-VH FTSYFIGWVRQMPGKGLEWMGIIYPGKSDTRYSPSFQGQVTISADKSISTAYLQW SSLKASDTAMYYCARVYSFGGRHKALF DYWGQGTLVTVSS55 PRT human GC5B596-VH QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLIN PYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVAL DYWGQGTLVTVSS 56 PRT human GC5B379, GC5B598,DIQMTQSPSSLSASVGDRVTITCRASQSI GC5B81 and SSYLNWYQQKPGKAPKLLIYAASSLQSGC5465-VL GVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQSYSTPLTFGQGTKVEIK 57 PRThuman GC5B373, GC5B376, EIVLTQSPATLSLSPGERATLSCRASQSV GC5B385, GC5B599,SSYLAWYQQKPGQAPRLLIYDASNRAT GC5B483-VL GEPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTKVEIK 58 PRT human GC5B596, GC5B602-DIQMTQSPSSLSASVGDRVTITCKASQN VL VATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDF ATYYCQQYNRYPYTFGQGTKLEIK 59 PRT humanIgG4PAA ASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK 60 PRT humanIgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLS PGK 61 PRT humanGC5B382-HCDR1 DYGMH 62 PRT human GC5B385-HCDR1 GYAMS 63 PRT humanGC5B370, GC5B597- SYGIS HCDR1 64 PRT human GC5B602-HCDR1 GYSFTGYTMN 65PRT human GC5B603-HCDR1 SYAMS 66 PRT human GC5B601-HCDR1 GFSLTSYNVH 67PRT human GC5B382-HCDR2 AIKYSGGSTYYADSVKG 68 PRT human GC5B603, GC5B385-AISGSGGSTYYADSVKG HCDR2 69 PRT human GC5B370, GC5B597- GIIPIFGNINYAQKFQGHCDR2 70 PRT human GC5B602-HCDR2 LINPYNGDTN 71 PRT human GC5B601-HCDR2VIWAGGSTNYNSALMS 72 PRT human GC5B382-HCDR3 RAESGPGLDY 73 PRT humanGC5B373-HCDR3 IGFYGRSFRIFDY 74 PRT human GC5B385-HCDR3 VDRSFGRSRYTLDY 75PRT human GC5B370, GC5B597- VSRRFKRFAYYFDY HCDR3 76 PRT humanGC5B603-HCDR3 SNFLPVVFDY 77 PRT human GC5B601-HCDR3 DGIRLRFAY 78 PRThuman GC5B382, GC5B370, WASTRES GC5B597-LCDR2 79 PRT human GC5B603-LCDR2TASNRAT 80 PRT human GC5B382, GC5B370, QQYYSTPLT GC5B597-LCDR3 81 PRThuman GC5B603-LCDR3 QQYFRAPIT 82 PRT human GC5B382-VHEVQLLESGGGLVQPGGSLRLSCAASGFT FSDYGMHWVRQAPGKGLEWVSAIKYSGGSTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAKRAESGPGLD YWGQGTLVTVSS 83PRT human GC5B379-VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVSGISYS GGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAAWDFGRR AVRLDYWGQGTLVTVSS 84 PRT human GC5B376-VHEVQLVQSGAEVKKPGESLKISCKGSGYS FTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQ WSSLKASDTAMYYCARIGFYGRSFRIF DYWGQGTLVTVSS85 PRT human GC5B376-VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG DSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVYSFGGRHKA LFDYWGQGTLVTVSS 86 PRT human GC5B385-VHEVQLLESGGGLVQPGGSLRLSCAASGFT FSGYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAKVDRSFGRSR YTLDYWGQGTLVTVSS87 PRT human GC5B370,  QVQLVQSGAEVKKPGSSVKVSCKASGG GC5B597-VHTFSSYGISWVRQAPGQGLEWMGGIIPIF GNINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARVSRRFKRFAYY FDYWGQGTLVTVSS 88 PRT human GC5B602-VHQVQLVQSGAEVKKPGASVKVSCKASG YSFTGYTMNWVRQAPGQGLEWMGLINPYNGDTNYAQKLQGRVTMTTDTSTSTA YMELRSLRSDDTAVYYCARVALRVAL DYWGQGTLVTVSS 89PRT human GC5B603-VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSG GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSNFLPVVFDY WGQGTLVTVSS 90 PRT human GC5B596-Atgcgggtgctggcccagctgctgggactgctgctgctgtgc light chainttccctggcgccagatgcgacatccagatgacccagagccccagcagcctgagcgccagcgtgggcgaccgggtgaccatcacctgcaaggccagccagaacgtggccacccacgtgggctggtaccagcagaagcccggcaaggcccccaagcggctgatctacagcgccagctaccggtacagcggcgtgcccagccggttcagcggcagcggcagcggcaccgagttcaccctgaccatcagcaacctgcagcccgaggacttcgccacctactactgccagcagtacaaccggtacccctacaccttcggccagggcaccaagctggagatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga 91 PRT human GC5B601-VHQVTLKESGPVLVKPTETLTLTCTVSGFS LTSYNVHWIRQPPGKALEWLAVIWAGGSTNYNSALMSRLTISKDTSKSQVVLT MTNMRAEDTATYYCARDGIRLRFAYW GQGTLVTVSS 92 PRThuman GC5B382, GC5B597, DIVMTQSPDSLAVSLGERATINCKSSQS GC5B370-VLVLYSSNNKNYLAWYQQKPGQPPKLLIY WASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGQGTK VEIK 93 PRT human GC5B603-VLEIVLTQSPATLSLSPGERATLSCRASQSV RKSLAWYQQKPGQAPRLLIYTASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAV YYCQQYFRAPITFGQGTKVEIKK 94 PRT humanGC5B601-VL EIVMTQSPATLSVSPGERATLSCKASQN VATHVGWYQQKPGQAPRLLIYSASYRYSGIPARFSGSGSGTEFTLTISSLQSEDFA VYYCQQYNRYPYTFGQGTKLEIK 95 PRT humanGC5B603-LCDR1 RASQSVRKSLA 96 DNA human GC5B596-heavyatggcctgggtctggaccctgctgttcctgatggccgctgccc chainagagcatccaggcccaggtgcagctggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaaggccagcggctacagcttcaccggctacaccatgaactgggtgcggcaggcccccggccagggcctggagtggatgggcctgatcaacccctacaacagcgacaccaactacgcccagaagctgcagggccgggtgaccatgaccaccgacaccagcaccagcaccgcctacatggagctgcggagcctgcggagcgacgacaccgccgtgtactactgcgcccgggtggccctgcgggtggccctggactactggggccagggcaccctggtgaccgtgagcagcgcctccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaaacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga

1-65. (canceled)
 66. An isolated GPRC5D×CD3 bispecific antibodycomprising: a) a first heavy chain (HC1); b) a second heavy chain (HC2);c) a first light chain (LC 1); and d) a second light chain (LC2),wherein the HC 1 and the LC 1 pair to form a first antigen-binding sitethat specifically binds CD3, and the HC2 and the LC2 pair to form asecond antigen-binding site that specifically binds GPRC5D; wherein HC1comprises an amino acid sequence of SEQ ID NO: 25, LC1 comprises anamino acid sequence of SEQ ID NO: 26, and wherein HC2 comprises an aminoacid sequence of SEQ ID NO: 55, and LC2 comprises an amino acid sequenceof SEQ ID NO:
 58. 67. The isolated GPRC5D×CD3 bispecific antibody ofclaim 66 wherein the antibody or bispecific binding fragment are ofIgG1, IgG2, IgG3, or IgG4 isotype.
 68. The isolated GPRC5D×CD3bispecific antibody of claim 66 wherein the antibody or bispecificbinding fragment is IgG4 isotype.
 69. The isolated GPRC5D×CD3 bispecificantibody of claim 66, wherein the GPRC5D binding portion of thebispecific antibody comprises a variable heavy (VH) chain regioncomprising the amino acid sequence of SEQ ID NO: 55, a variable light(VL) chain region comprising the amino acid sequence of SEQ ID NO: 58,and an Fc domain comprising the amino acid sequence of SEQ ID NO: 59.70. The isolated GPRC5D×CD3 bispecific antibody of claim 66, wherein theCD3 binding portion of the bispecific antibody comprises a variableheavy (VH) chain region comprising the amino acid sequence of SEQ ID NO:25, a variable light (VL) chain region comprising the amino acidsequence of SEQ ID NO: 26, and an Fc domain comprising the amino acidsequence of SEQ ID NO: 59, wherein the Fc domain further comprises aF405L and a R409K substitution.
 71. The isolated GPRC5D×CD3 bispecificantibody of claim 66, wherein the antibody or bispecific bindingfragment thereof binds GPRC5D on the surface of human myeloma cells. 72.The isolated GPRC5D×CD3 bispecific antibody of claim 66, wherein theantibody or bispecific binding fragment thereof binds GPRC5D on thesurface of human multiple myeloma cells.
 73. The isolated GPRC5D×CD3bispecific antibody of claim 66, wherein the antibody or bispecificbinding fragment induces human T cell activation in vitro with an EC₅₀of less than about 0.22 nM.
 74. The isolated GPRC5D×CD3 bispecificantibody of claim 66, wherein the antibody or bispecific bindingfragment induces T-cell dependent cytotoxicity of GPRC5D-expressingcells in vitro with an EC₅₀ of less than about 0.89 nM.
 75. An isolatedcell expressing the isolated GPRC5D×CD3 antibody of claim
 66. 76. Theisolated cell of claim 75, wherein the cell is a hybridoma.
 77. Theisolated cell of claim 75, wherein the GPRC5D×CD3 antibody isrecombinantly produced.
 78. A method for treating a subject havingcancer, the method comprising: administering a therapeutically effectiveamount of the isolated GPRC5D×CD3 bispecific antibody of claim 66 to apatient in need thereof for a time sufficient to treat the cancer.
 79. Amethod for inhibiting growth or proliferation of cancer cells, themethod comprising: administering a therapeutically effective amount ofthe isolated GPRC5D×CD3 bispecific antibody of claim 66 for a timesufficient to inhibit the growth or proliferation of cancer cells.
 80. Amethod of redirecting a T cell to a GPRC5D-expressing cancer cell, themethod comprising: administering a therapeutically effective amount ofthe isolated GPRC5D×CD3 bispecific antibody of claim 66 for a timesufficient to redirect a T cell to a cancer.
 81. The method of claim 78wherein the cancer is a hematological cancer.
 82. The method of claim 81wherein the hematological cancer is a GPRC5D-expressing B cell cancer.83. The method of claim 82 wherein the GPRC5D-expressing B cell canceris multiple myeloma.
 84. The method of claim 78, wherein the methodfurther comprises administering a second therapeutic agent.
 85. Themethod of claim 84, wherein the second therapeutic agent is achemotherapeutic agent or a targeted anti-cancer therapy.
 86. The methodof claim 85, wherein the chemotherapeutic agent is cytarabine, ananthracycline, histamine dihydrochloride, or interleukin
 2. 87. Themethod of claim 85, wherein the second therapeutic agent is administeredto said subject simultaneously with, sequentially, or separately fromthe bispecific antibody.
 88. A pharmaceutical composition comprising theisolated GPRC5D×CD3 bispecific antibody of claim 66 and apharmaceutically acceptable carrier.
 89. A method for generating theisolated GPRC5D×CD3 bispecific antibody of claim 66, the methodcomprising culturing a cell expressing the isolated GPRC5D×CD3 antibodyunder conditions sufficient to produce the bispecific antibody.
 90. Anisolated synthetic polynucleotide encoding the isolated GPRC5D×CD3bispecific antibody of claim
 66. 91. A kit comprising the isolatedGPRC5D×CD3 bispecific antibody of claim 66 and packaging for the same.92. A kit comprising the isolated synthetic polynucleotide of claim 90and packaging for the same.